Silane-treated silica filter media

ABSTRACT

The present invention provides methods for separating one or more components of interest from a sample containing particulates and soluble materials. The method comprises the steps of: (a) filtering a sample through silica filter media whose surface silanol groups have reacted with one or more silanes, and (b) simultaneously capturing particulates and binding a soluble component to the silica filter media. The bound soluble component of interest is subsequently eluted from the silica filter media. In one embodiment of the invention, unwanted soluble materials are captured by the treated silica filter media and desired component of interest is recovered from the flow-through. In another embodiment, different components of interest are recovered from both the eluate and the flow-through. Preferred treated silica filter media are silane-treated rice hull ash or diatomaceous earth with functional quarternary ammonium group or functional sulphonate group. The present invention also provides silane-treated silica filter media.

This application is a continuation of U.S. application Ser. No.11/828,275, filed Jul. 25, 2007, now U.S. Pat. No. 7,374,684; which is acontinuation of U.S. application Ser. No. 10/830,935, filed Apr. 23,2004, now U.S. Pat. No. 7,264,728; which is a continuation-in-part ofU.S. application Ser. No. 10/677,404, filed Oct. 1, 2003, abandoned;which claims the benefit of provisional application 60/415,474, filedOct. 1, 2002. The above-identified applications are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods for separating one or morecomponents of interest from a sample containing particulate matter andsoluble components. More particularly, the invention relates to the useof silane-treated silica filter media such as rice hull ash forseparating protein and capturing particulates simultaneously. Examplesof particulates include microorganisms.

BACKGROUND OF THE INVENTION

The production of materials in biotechnology involves the isolation,separation, and purification of a specific material that is surroundedby many other biological components. It does not matter whether thematerial comes from fermentation, a transgenic plant or the milk of atransgenic goat; the material of interest must be collected in areasonably pure form. When the starting mixture is very complex,isolation of the material of interest can be especially difficult andoften requires costly operations. Technologies that reduce the number ofseparation operations and simplify recovery procedures are in highdemand in biotechnology and several other industries including watertreatment, food and beverage, and chemicals.

Separation of product from microorganisms is important because microbialcontamination is a common problem across many industries, includingbrewing, winery, juice and beverages, dairy, industrial enzyme andpharmaceutical. Heat sterilization and size-based filtration are by farthe most commonly used processes to address this. Each of these methodshas its advantages and disadvantages. The main drawback of heatsterilization is its application is limited to products that are notaffected by high temperature. Sized-based filtration has thedisadvantages of being expensive and time consuming. In addition, itcannot be used for processes in which the desired components are of thesame size as bacteria, such as in the dairy food industry.

Examples of technologies that have been developed to simplifyseparations include Expanded Bed Adsorption and Chromatography. ExpandedBed Adsorption allows the capture of a soluble component from afermentation mixture containing both soluble and particulate components.This method does not require a pre-filtration step prior to applying thesample to the bed. The fermentation mixture flows upward through a bedof adsorbent beads; the upward flow lifts and suspends the beads as thebed expands upward. The soluble components are captured by the beadswhile the particulate matter flows around the beads and exits the top ofthe bed. Then the soluble components are recovered from the beads by anelution step. This technology is not widely used yet as there areseveral technical hurdles including scale-up difficulty, maintaining astable bed, carry-over of beads out of the top of the bed, fouling ofthe beads by the fermentation mixture, cleaning and re-use of the beads,usable life of the beads, and variable pressure drop during the courseof the adsorption step.

Solid-Liquid Chromatography is any separation process that depends onsolute(s) partitioning between a flowing fluid and a solid adsorbent.Many different solid adsorbents (generally referred to as“stationary-phase packing”) are used in chromatography. Differentstationary-phase packings give rise to different chromatographictechniques, which are generally classified according to their mechanismof interactions. The interactions could be through one or more of thefollowing mechanisms: charge (ion-exchange chromatography); van derWaals forces (hydrophobic interaction chromatography); size and shape(size exclusion); affinity (for example, protein-A, biotin-avidin,biotin-streptavidin, lectin, antibodies, pectin, dye ligand, immobilizedmetal affinity) (Reference: “Biochemical Engineering” by Harvey W.Blanch and Douglas S. Clark, Marcel Dekker Inc, 1996; p 502-506). CustomAffinity Chromatography is designed to capture a specific protein andrequires a specific affinity medium with a specific ligand for eachprotein to be captured. Considerable time, effort, and cost are involvedin developing this specific medium. In general, chromatography requiresa pre-filtration step to remove solid materials.

Filtration is the removal of particulates by passing a feed streamthrough a porous media. Particulates are captured on the media through avariety of mechanisms including direct impaction, sieving, and others.Filtration methods employing various types of media have been used toremove particulates in such applications as wastewater treatment,winemaking, beverage making, and industrial enzyme production.

Filter media, also known as filter aids, can be loose particulate orstructured material. They are solid materials in a particulate form,insoluble in the liquid to be filtered; they are added to the liquid orare coated upon a filter or filter support. The purpose of using filtermedia is to speed up filtration, reduce fouling of the filter surface,reduce cracking of the filter layer, or otherwise to improve filtrationcharacteristics. Materials, which are frequently used as filter media,include cellulose fibers, diatomaceous earth, charcoal, expandedperlite, asbestos fibers and the like.

Filter media are often described according to their physical form. Somefilter media are essentially discrete membranes, which function byretaining contaminants upon the surface of the membrane (surfacefilters). These filter media primarily operate via mechanical straining,and it is necessary that the pore size of the filter medium be smallerthan the particle size of the contaminants that are to be removed fromthe fluid. Such a filter medium normally exhibits low flow rates and atendency to clog rapidly.

Other filter media take the form of a porous cake or bed of fine fibrousor particulate material deposited on a porous support or substrate. Thesolution being filtered must wend its way through a path of pores formedin the interstices of the fine material, leaving particulatecontaminants to be retained by the filter material. Because of thedeepness of the filter material, the filters are called depth filters(as opposed to surface filters). Depth filters typically retaincontaminants by both the sieving mechanism and the electrokineticparticle capture mechanism. In the electrokinetic particle capture mode,it is unnecessary that the filter medium have such a small pore size.The ability to achieve the required removal of suspended particulatecontaminants with a filter medium of significantly larger pore size isattractive inasmuch as it allows higher flow rates. Furthermore, thefilters have a higher capacity to retain particulates, thus having areduced tendency to clog.

Biotechnology and other industries need efficient, cost-effectivemethods to isolate components of interest. It is also desirable to uselow-cost raw materials for the process of separating biomolecules.

Rice hull ash is a byproduct of rice farming and rice is a primary foodstaple for half of the world's population. Currently, the inedible ricehulls are used as a source of fuel, fertilizer, and in insulationapplications. When rice hulls are burned, a structured particle materialhaving free acidic hydroxyl moieties (OH or Particle-OH) on the surfacemuch like particle-OH of precipitated silica or fumed silica can beproduced as a byproduct that has been demonstrated to be useful as afilter aid. U.S. Pat. No. 4,645,605 discloses the use of rice hull ashas filtration media.

U.S. Pat. No. 4,645,567 discloses that the filtration of fine particlesize contaminants from fluids has been accomplished by the use ofvarious porous filter media through which the contaminant fluid ispassed. To function as a filter, filter media must allow the fluid(commonly water) through, while holding back the particulate. Thisholding back of the particulate is accomplished by distinctly differentfiltration mechanisms, namely (a) mechanical straining and (b) particlecapturing. In mechanical straining, a particle is removed by physicalentrapment when it attempts to pass through a pore smaller than itself.In particle capturing, the particle collides with a surface face withinthe porous filter media and is retained on the surface by short-rangeattractive forces.

WO 02/083270 discloses a filter system for passive filtration. Thesystem comprising: a housing with an intake and an outlet; a pleatedcarbon filter disposed between the intake and the outlet for filteringout vapors entering the intake; and a hydrophobic solution including asilane composition dispersed about the pleated carbon filter to inhibitadsorption of water thereby increasing the adsorption capacity of thepleated carbon filter especially in high relative humidity environmentsand wherein the hydrophobic solution is selected so that it does notdecrease the adsorption capacity of the carbon filter.

U.S. Pat. No. 6,524,489 discloses advanced composite media comprisingheterogeneous media particles, each of said media particles comprising:(i) a functional component selected from the group consisting ofdiatomite, expanded perlite, pumice, obsidian, pitchstone, and volcanicash; and (ii) a matrix component selected from the group consisting ofglasses, natural and synthetic crystalline minerals, thermoplastics,thermoset plastics with thermoplastic behavior, rice hull ash, andsponge spicules; wherein said matrix component has a softening pointtemperature less than the softening point temperature of said functionalcomponent; and wherein said functional component is intimately anddirectly bound to said matrix component. The surface of the advancedcomposite media can be treated with dimethyldichlorosilane,hexamethyldisilazane, or aminopropyltriethoxysilane.

Snyder, et al. disclose chromatography bonded-phase packing prepared bythe reaction of organosilanols, organodimethylamine, or organoalkoxysilanes with high surface area silica supports without polymerization.(L. R. Snyder and J. J. Kirkland, Introduction to Modern LiquidChromatography, 2nd edition, Wiley-Interscience, N.Y. 1979. 272-280)

Roy, et al (J. Chrom. Sci. 22: 84-86 (1984)) disclose the preparation ofion-exchange (DEAE, carboxy, and sulfonic) silica using the epoxyfunctionality of glycidoxypropylsilyl silica; the ion-exchange silicawas used in column chromatography to separated bovine serum albumin andbovine γ-globulin.

In general, treated chromatographic silica of the type described bySnyder and Roy are too expensive to be used in larger scale routinefiltration and isolation processes.

There is a need for an improved and less costly separation system thatis suitable for large-scale isolation of components of interest from asample. Such a system uses low-cost raw materials and is suitable for alarge-scale production and requires no pretreatment of a sample.

SUMMARY OF THE INVENTION

The present invention provides methods for separating one or morecomponents, especially biomolecules of interest, from a samplecontaining particulates and soluble materials. The feature of theinvention is filtering a sample through filter media whose surface hasbeen treated by one or more silanes. Preferred filter media are silicafilter media. The methods provide simultaneously capturing theparticulate by filtration and binding soluble materials onto the silicafilter media.

One method of the invention comprises the steps of: (a) filtering asample through the treated silica filter media, (b) simultaneouslycapturing particulates and binding a soluble component of interest tothe silica filter media, and (c) eluting the bound soluble component ofinterest from the silica filter media.

Another method of the invention comprises the steps of: (a) filtering asample through treated silica filter media, (b) simultaneously capturingparticulates and binding unwanted soluble materials to the silica filtermedia, (c) collecting the flow-through stream, and (d) recovering thesoluble component of interest from the flow-through stream.

Another method of the invention comprises the steps of: (a) filtering asample through treated silica filter media; (b) simultaneously removingparticulate and binding a first soluble component of interest to thesilica filter media, (c) collecting the flow-through stream, (d)recovering a second soluble component of interest from the flow-throughstream, (e) eluting the bound first soluble component of interest fromthe silica filter media, and (f) recovering the first soluble componentof interest.

In one embodiment of the invention, the particulates are microorganisms.In addition to being captured by the silane-treated filter media,microorganisms are also found killed by contacting with thesilane-treated filter media.

The present invention is also directed to the silane-treated filtermedia. Preferred treated silica filter media are silane-treated ricehull ash with a functional quaternary ammonium group(s) or a functionalsulphonate group(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows protein binding, and FIG. 1B shows protein release, tountreated diatomaceous earth (FW12), untreated rice hull ash, HQ50(commercial quaternary amine anion exchange resin) and surface treatedrice hull ashes (silica filter media samples 4 and 6).

FIG. 2 shows protein binding and protein release using surface-treatedrice hull ashes. FIG. 2A shows the results of silica filter mediasamples 7 and 8. FIG. 2B shows the results of samples 9 and 10. FIG. 2Cshows the results of samples 11 and 12.

FIG. 3 shows protein binding and protein release using surface-treatedrice hull ashes. FIG. 3A shows the results of sample 14. FIG. 3B showsthe results of silica filter media samples 13 and 15. FIG. 3C shows theresults of samples 16 and 17. FIG. 3D shows the results of samples 18and 19. FIG. 3E shows the results of sample 20.

FIG. 4 shows protein binding and protein release using surface-treatedrice hull ashes. FIG. 4A shows the results of silica filter media sample41 and untreated RHA. FIG. 4B shows the results of porous HS50.

FIG. 5 shows protein binding and protein release. FIG. 5A shows theresults of silica filter media sample 42. FIG. 5B shows the results ofsample 40 and untreated RHA. FIG. 5C shows the results of Celite 512.FIG. 5D shows the results of sample 29 and untreated RHA.

FIG. 6 shows dynamic protein binding and protein release usingsurface-treated RHA (sample 9).

FIG. 7A shows untreated rice hull ash, and FIG. 7B shows silica filtermedia sample 19, for simultaneous particulate filtration and solublecapture/release.

FIG. 8 shows silica filter media sample 19 and untreated RHA forsimultaneous particulate filtration and soluble capture release.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for separating one or morecomponents of interest from a sample. One embodiment of the inventioncomprises the steps of: (a) filtering a sample containing particulateand soluble components through silica filter media whose surface hasbeen treated with one or more silanes, (b) simultaneously capturingparticulates and binding a soluble biomolecule of interest to the silicafilter media, and (c) eluting the bound soluble component of interestfrom the silica filter media. In this embodiment, the molecule ofinterest is first bound to the silica filter media and recovered laterby elution. Optionally, an insoluble component of interest can berecovered from the particulates.

Another embodiment of the invention comprises the steps of: (a)filtering a sample containing particulate and soluble materials throughsilica filter media whose surface has been treated with one or moresilanes, (b) simultaneously capturing particulates and binding unwantedsoluble materials to the silica filter media, (c) collecting theflow-through stream, and (d) recovering the soluble component ofinterest from the flow-through stream. The soluble component of interestcan be further purified from the flow-through stream. In thisembodiment, the soluble component of interest does not bind to thesilica filter media and is recovered in the flow-through. Optionally, aninsoluble component of interest can be recovered from the particulates.

Yet another embodiment of the invention comprises the steps of: (a)filtering a sample containing particulate and soluble materials throughsilica filter media whose surface has been treated with one or moresilanes; (b) simultaneously capturing particulates and binding a firstsoluble component of interest to the silica filter media, (c) collectingthe flow-through stream, (d) recovering a second soluble component ofinterest from the flow-through stream, (e) eluting the bound firstsoluble component of interest from the silica filter media, and (f)recovering the first soluble component of interest. In this embodiment,the first component of interest binds to the silica filter media and thesecond component of interest does not bind to the silica filter media.Optionally, an insoluble component of interest can be recovered from theparticulates.

The present invention optionally comprises an additional step. Prior tothe filtering step (step (a)), a sample containing particulate andsoluble materials first reacts with the treated silica filter media fora period of time to allow sufficient binding of the component to surfaceof the treated silica filter media. The reaction is carried out bymixing the sample with the treated silica filter media by any means ofmechanical mixing such as agitation, stirring, vortexing, etc. After thecomponent binds to the treated silica filter media, the mixture isapplied to a filtration device and the sample is subsequently filteredthrough the filter media.

The term “sample” refers to any mixture containing multiple componentsin the form of a liquid, solution, suspension or emulsion. The sampleusually includes soluble components and particulates. Of specialinterest are “biological samples” which refers to biological tissueand/or fluid that contains biomolecules such as polypeptides, lipids,carbohydrates, lipoproteins, polysaccharides, sugars, fatty acids,polynucleotides, or viruses. A biological sample may contain sections oftissues such as frozen sections taken for histological purposes. Asample suitable for this invention includes cell lysate, culture broth,food products and particularly dairy products, blood, beverages (forexample, juice, beer, wine), and a solution or a suspension containingbiomolecules such as proteins. “Proteins” are natural, synthetic, andengineered peptides or polypeptides, which include enzymes such asoxidoreductases, transferases, isomerases, ligases, and hydrolases,antibodies, hormones, cytokines, growth factors, and other biologicalmodulators.

Filtration is the removal of particulates by passing a feed streamthrough a porous media. Particulates are captured on the media through avariety of mechanisms including physical entrapment, and binding to themedia. The present invention utilizes silica media filter of varioustypes to remove particulates in different applications, includingwastewater treatment, winemaking, juice and beverage making, diary, andindustrial production of proteins such as enzymes.

The term “particulates” refers to macroscopic insolubles or microscopicparticulates. Macroscopic particulates are those that are visible to thehuman eye, including, but not limited to precipitates, inclusion bodies,and crystals. Inclusion bodies consist of insoluble and incorrectlyfolded protein in the cellular compartment. Crystals are formed fromsupersaturated solutions by aggregation of molecules, occurring in anordered, repetitive fashion. Precipitates are amorphous form from randomaggregation. Macroscopic particulates can be of organic or inorganicorigin; they can be derived from the interaction between protein andprotein, salt and protein, salt and salt, protein and polymer, etc.Microscopic particulates are those that can be seen under a microscope.Examples of particulates include microorganisms. Microorganisms suitableto be captured and removed from a biological sample by the presentinvention are gram-positive bacteria, gram-negative bacteria, fungi,yeast, mold, virus, etc.

The feature of this invention is using treated silica filter media in afiltration process to simultaneously bind soluble components onto thesilica filter media and capture particulates from a solution byfiltration. The present invention eliminates a pre-filtration step oftenrequired in a chromatography process to remove particulate. Solublecomponents bind to the silane-treated silica filter media throughdifferent mechanisms such as hydrophilic, hydrophobic, affinity and/orelectrostatic interactions. Silica filter media useful for thisinvention have surfaces suitable for treatment with silanes andstructural characteristics suitable for industrial filtrationapplications. Examples of silica filter media include, but are notlimited to, rice hull ash, oat hull ash, diatomaceous earth, perlite,talc, and clay.

Rice hull ash is a byproduct of rice farming. Each grain of rice isprotected with an outer hull, which accounts for 17-24% of the roughweight of the harvested product. Rice hulls consist of 71-87% (w/w)organic materials, such as cellulose and 13-29% (w/w) inorganicmaterials. A significant portion of the inorganic fraction, 87-97% (w/w)is silica (SiO₂). Currently, the inedible rice hulls are used as asource of fuel, fertilizer, and in insulation applications. When therice hulls are burned, a structured silica material (often greater than90%) can be produced as a byproduct. Rice hull ash (RHA) has largersurface area and more porous-channeled structure compared with otherloose silica filter media. These characteristics make the RHA apreferred treated filter substrate for this invention.

Diatomaceous earth (Diatomite) is a sedimentary silica deposit, composedof the fossilized skeletons of diatoms, one celled algae-like plantswhich accumulate in marine or fresh water environments. The honeycombsilica structures give diatomite useful characteristics such asabsorptive capacity and surface area, chemical stability, and low bulkdensity. Diatomite contains 90% SiO₂ plus Al, Fe, Ca and Mg oxides.

Perlite is a generic term for a naturally occurring siliceous volcanicrock that can be expanded with heat treatment. Expanded perlite can bemanufactured to weigh as little as 2 pounds per cubic foot (32 kg/m³).Since perlite is a form of natural glass, it is classified as chemicallyinert and has a pH of approximately 7. Perlite consists of silica,aluminum, potassium oxide, sodium oxide, iron, calcium oxide, andmagnesium oxide. After milling, perlite has a porous structure that issuitable for filtration of coarse microparticulates from liquids it issuitable for depth filtration.

Talc (talcum) is a natural hydrous magnesium silicate, 3 MgO.4SiO₂.H₂O.Clay is hydrated aluminum silicate, Al₂O₃.SiO₂.xH₂O. Mixtures of theabove silica filter media substrates can also be used to achieve thebest filtration and cost performance. The rice hull ash or diatomaceousearth has optionally undergone various purification and/or leachingsteps before the surface silane treatment.

Silica filter media are treated by binding a predetermined amount offunctional silane (or silanes) to the surface. The treated silica filtermedia capture components, for example, by electrostatic, hydrophilic,hydrophobic, affinity interactions, and/or by physical entrapment. Byelectrostatic interaction, the charged silica filter media bind tomaterials in a sample that have the opposite charge. By hydrophilicinteraction, the portion of the silica filter media that has a strongaffinity for water attracts the polar group of the materials by van derWaals interaction. By hydrophobic interaction, the portion of the silicafilter media that contains long hydrocarbon chains attracts thenon-polar groups of the materials. The treated silica filter mediaselectively capture materials (desired or undesired) during theseparation process, which results in better separation characteristicscomparing with non-treated silica filter media. The treated silicafilter media preferably have a similar or improved flow rate comparedwith the non-treated silica filter media.

The form of silica filter media substrate materials can be any formsuitable for the application, such as spheres, fibers, filaments,sheets, slabs, discs, blocks, films, and others. They can bemanufactured into cartridges, disks, plates, membranes, woven materials,screens etc. The specific surface area of the untreated silica filtermedia is preferred to be larger than 1 m²/g; more preferred to be largerthan 10 m²/g. Silica filter media with a larger surface area arepreferable because they allow more treatment on the surface. Inaddition, media with large pores improve the filtration rate. However,larger pore materials have relatively lower surface area. The balance oflarge surface area and large pore size results in effective surfacefiltration treatment and filtration rate. The surface characteristics ofthese substrates can be evaluated by techniques such as NMR (NuclearMagnetic Resonance and other techniques), SEM (Scanning ElectronMicroscopy), BET (Brunauer-Emmett-Teller) surface area measurementtechnique, and Carbon-hydrogen-nitrogen content can be determined bycombustion techniques, which are well known to the art.

Silanes suitable for surface treatment of silica filter media can be anytype of organosilanes, ionic or non-ionic. The general formula of thesuitable silane is (R¹)_(x)Si(R²)_(3-x)R³,

wherein R¹ is typically a hydrolysable moiety (such as alkoxy, halogen,hydroxy, aryloxy, amino, amide, methacrylate, mercapto, carbonyl,urethane, pyrrole, carboxy, cyano, aminoacyl, or acylamino, alkyl ester,or aryl ester), which reacts with the active group on the silica filtermedia; a preferred hydrolysable moiety is alkoxy group, for example, amethoxy or an ethoxy group;

1≦x≦3, more than one siloxane bond can be formed between the filterparticle and silane;

R² can be any carbon-bearing moiety that does not react with the filtersurface during treatment process, such as substituted or unsubstitutedalkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl,heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl,alkcycloalkaryl, alkcycloalkenyaryl, or arylalkaryl;

R³ can be any organic containing moiety that remains chemically attachedto the silicon atom once the surface reaction is completed, andpreferably it can interact with the component of interest duringfiltration; for example R₃ is hydrogen, alkyl, alkenyl, alkaryl,alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic,cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl,arylakaryl, alkoxy, halogen, hydroxy, aryloxy, amino, amide,methacrylate, mercapto, carbonyl, urethane, pyrrole, alkyl ester, arylester, carboxy, sulphonate, cyano, aminoacyl, acylamino, epoxy,phosphonate, isothiouronium, thiouronium, alkylamino, quaternaryammonium, trialkylammonium, alkyl epoxy, alkyl urea, alkyl imidazole, oralkylisothiouronium;

wherein the hydrogen of said alkyl, alkenyl, aryl, cycloalkyl,cycloalkenyl, heteroaryl, and heterocyclic is optionally substituted byhalogen, hydroxy, amino, carboxy, or cyano.

One or more silanes can be covalently bound to the surface of thehydroxyl bearing porous silica filter media. The surface area of thesilica filter media limits the amount of the silanes bound.

Silanes useful for treating silica in this invention preferably have oneor more moieties selected from the group consisting of alkoxy,quaternary ammonium, aryl, epoxy, amino, urea, methacrylate, imidazole,carboxy, carbonyl, isocyano, isothiorium, ether, phosphonate, sulfonate,urethane, ureido, sulfhydryl, carboxylate, amide, carbonyl, pyrrole, andionic. Examples for silanes having an alkoxy moiety are mono-, di-, ortrialkoxysilanes.

Examples of silanes having a quaternary ammonium moiety are3-(trimethoxysilyl)propyloctadecyldimethylammoniumchloride,N-trimethoxysilylpropyl-N,N,N-trimethylammoniumchloride, or3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilanehydrochloride. Examples of silanes having an aryl moiety are3-(trimethoxysilyl)-2-(p,m-chlandomethyl)-phenylethane,2-hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone,((chloromethyl)phenylethyl)trimethoxysilane andphenyldimethylethoxysilane. Examples of silanes having an epoxy moietyare 3-glycidoxypropyltrimethoxysilane and2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane. Examples of silanes havingan amino moiety are 3-aminopropyltrimethoxysilane,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,trimethoxysilylpropyldiethylenetriamine,2-(trimethoxysilylethyl)pyridine, N-(3-trimethoxysilylpropyl)pyrrole,trimethoxysilylpropyl polyethyleneimine,bis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane, andbis(2-hydroxyethyl)-3-aminopropyltriethoxysilane. Examples of silaneshaving an urea moiety are N-(triethoxysilylpropyl)urea andN-1-phenylethyl-N′-triethoxysilylpropylurea. An example of silaneshaving a methacrylate moiety is 3-(trimethoxysilyl)propyl methacrylate.An example of silanes having a sulfhydryl moiety is3-mercaptopropyltriethoxysilane. Examples of silanes having an imidazolemoiety are N-[3-(triethoxysilyl)propyl]imidazole andN-(3-triethoxysilylpropyl)-4,5-dihydroimidazole. Examples of ionicsilanes are 3-(trimethoxysilyl)propyl-ethylenediamine triacetic acidtrisodium salt; and 3-(trihydroxysilyl)propylmethylposphonate sodiumsalt. An examples of silanes having a carbonyl moiety is3-(triethoxysilyl)propylsuccinic anhydride. Examples of silanes havingan isocyano moiety are tris(3-trimethoxysilylpropyl)isocyanurate and3-isocyanatopropyltriethoxysilane. Examples of silanes having an ethermoiety are bis[(3-methyldimethoxysilyl)propyl]-polypropylene oxide andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane.

An example of a silane having a sulfonate moiety is2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane. An example of a silanehaving a isothiourium moiety is trimethoxysilylpropylisothiouroniumchloride. Examples of silanes having an amide moiety aretriethoxysilylpropylethyl-carbamate,N-(3-triethoxysilylpropyl)-gluconamide, andN-(triethoxysilylpropyl)-4-hydroxybutyramide. Examples of silanes havinga urethane moiety are N-(triethoxysilylpropyl)-O-polyethylene oxideurethane and O-(propargyloxy)-N-(triethoxysilylpropyl)urethane.

Silica filter media can also be treated with more than one silanes suchas 3-aminopropyltrimethoxysilane andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane;3-trihydrosilylpropylmethylphosphonate, sodium salt andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane;N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride and(3-glycidoxypropyl)trimethoxysilane;3-trihydrosilylpropylmethylphosphonate, sodium salt andbis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane;3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilanehydrochloride and N-(triethoxysilylpropyl)-O-polyethylene oxideurethane; 2-(trimethoxysilylethyl)pyridine andN-(3-triethoxysilylpropyl)-gluconamide;N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride andN-(3-triethoxysilylpropyl)-gluconamide;N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride and2-hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone;3-mercaptopropyltriethoxysilane andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane;3-(triethoxysilyl)propylsuccinic anhydride andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane;trimethoxysilylpropyl-ethylenediamine, triacetic acid, trisodium saltand N-(triethoxysilylpropyl)-O-polyethylene oxide urethane;2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane; and2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane andbis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane.

The silane-treated silica filter media have a general formula selectedfrom the group consisting of particle-O—Si(R¹)_(x)(R²)_(3-x)R³,

wherein R¹, R², R³, and x are the same as described above so long asthere are no more than four groups directly attached to the silicon(Si);

R⁵, R⁶, R⁸ are independently hydrogen, substituted or unsubstitutedalkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl,heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl,alkcycloalkaryl, alkcycloalkenyaryl, ether, ester or arylalkaryl;

R⁴, R⁷, R⁹ are substituted or unsubstituted alkyl, alkenyl, alkaryl,alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic,cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, orarylalkaryl radicals capable of forming two covalent attachments.

The silica filter media with surface silanol are treated with silane ina general reaction scheme as following:Particle-OH+(R¹)_(x)Si(R²)_(3-x)R³→Particle-O—Si(R¹)_(3-n)(R²)_(3-x)R³+nR¹Hwhere Particle-OH is a filter particle with reactive sites on surface.For example, R¹ is a methoxy (CH₃O—) or ethoxy (CH₃CH₂O—) labile leavinggroup of the silane, which chemically interacts, with the reactivehydroxyl group on the particle surface or with other reactive hydrolyzedsilane molecules which are not attached to the surface. 1≦x≦3; n is thenumber of R¹ groups reacted, and n≦x.

Prolonged reaction of excess amounts of reactive silane under anhydrousconditions results in reaction of only 25% to 50% of the total activesites on the porous material since further reaction is inhibited bysteric hindrance between the immobilized residues and is also hinderedby access to deeply imbedded Particle-OH groups. For the purposes ofthis invention, such sterically available sites will be designated asthe “saturation coverage” and “saturation coverage” depends upon thesteric requirements of a particular residue. Note that this designationof “saturation coverage” is applicable to reactive silanes with one ormore labile leaving groups. Under anhydrous conditions, such silanesform monolayers and cannot form multiple layers of undefined saturation.However, under aqueous conditions, multiple layers can be built on thesurface with multifunctional silanes.

The surface silane treatment of silica filter media can be carried outby an essentially “wet” or essentially “dry” process. The essentiallywet process consists of reacting the silane onto the silica filter mediain a solvent (organic solvent or water) and optionally using heat. Heator solvent is not required for the reaction; however, heat or solventimproves the reaction rate and the uniform surface coverage. Theessentially dry process consists of reacting the silane onto the silicafilter media in a vapor phase or highly stirred liquid phase by directlymixing the silane with silica filter media and subsequently heating.

A preferred method for treating silica filter media with silanes isadding the reacting silanes gradually to a rapidly stirred solvent,which is in direct contact with the porous silica filter media. Anotherpreferred method is to carry out the treatment in the vapor phase bycausing the vapor of the reactive silanes to contact and react with thesilica filter media. For example, the porous material is placed in avacuum reactor and dried under vacuum. The rapidly reacting silane isthen allowed to enter the vacuum chamber as a vapor and contact theporous material; after a certain contact time, the byproducts of thereaction are removed under reduced pressure. Then the vacuum isreleased, and the porous material is removed from the chamber.

The actual treatment process can be carried out in a period from 1minute to 24 hours. Generally, for purposes of this invention, it ispreferred to carry out the treatment over a period from about 30 minutesto 6 hours to ensure that the surface of the filter aid material isuniformly treated. The treatments are carried out at temperaturesranging from 0 to 400° C. Preferred treatment temperatures are from roomtemperature (22 to 28° C.) to 200°.

The amount of reacting silanes used in this invention depends on thenumber of surface hydroxyls to be reacted, and the molecular weight ofthe silane. Typically, a stoichiometric amount equivalent to theavailable surface hydroxyls plus some excess amount of the reactingsilane is used to treat the surface hydroxyls because of the potentialside reactions. If a thicker exterior surface treatment is desired, thenmore reacting silane should be used. Typically, 0 to 10 (preferred), 0to 20, or 1 to 50 times excess is used. However, it is not uncommon touse from 1 to 500 times excess; which results in more treatment on theparticle.

Silanes with hydrolysable groups condense with Particle-OH groups of thesurface of the particles, and provide covalent coupling of organicgroups to these substrates. For example, the alkoxy groups of thesilanes chemically react with the Particle-OH groups of the particlesurface. The surface-silane interaction is fast and efficient. Forexample, when silanes having a quaternary ammonium moiety are used, theprotonated positively charged silanes electrostatically attract to thedeprotonated groups of the particle efficiently to facilitate fast andefficient reaction.

Silane-reacted silica filter media preferably have a functional moiety,which can react with a component of interest. The functional moiety isselected from the group consisting of quaternary ammonium, epoxy, amino,urea, methacrylate, imidazole, sulphonate and other organic moietiesknown to react with biological molecules. In addition, the functionallymoiety can be further reacted, using well-known methods, to createfurther new functionalities for other interactions. General schemes forpreparation of a silane-reacted particle filter media with a functionalquaternary ammonium or sulphonate group are illustrated as follows.

Silane-reacted particle filter media with a functional quaternaryammonium group can be prepared in one step. Optionally, a two step orthree step process may be employed. For example, in the first step ofthe two step process, the particle surface is reacted with anamino-functional silane, (R¹)_(X)Si(R²)_(3-X)R⁴N(R⁵)₂, applying thepreviously described procedure. In the next step, the secondary aminereadily reacts with the epoxide group of theglycidyltrimethylammoniumchloride, which is a convenient way tointroduce quaternary ammonium functionality. (See Scheme 1)

Silane-reacted silica filter media with a functional sulphonate groupcan be prepared in two steps. In the first step, the particle surface isreacted with an epoxy-functional silane applying the previouslydescribed procedure. In the next step, the epoxy functionality readilyreacts with sodium bisulfate to produce sulphonate-functional silicafilter media. (See Scheme 2). Sodium metabisulfite (Na₂S₂O₅) decomposesin water to form sodium bisulfate (NaHSO₃).

The silane-treated particles are used in separation applications tocapture soluble materials through electrostatic, and/or hydrophobic,and/or hydrophilic interaction mechanisms while removing particulates.The advantage of the treated silica filter media is that the separationprocess is simplified by combining the filtration and solid phaseextraction in a single step. The desired quality of the treated silicafilter media is to have similar or improved flow rate (filtrationproperties) to the untreated silica filter media along with thecapability to capture soluble materials through sorption in a singleoperation.

In one embodiment of the invention, specific charged groups are attachedcovalently to the surface of the silica particles to capture materialselectrostatically. The oppositely charged materials are bound to theporous treated surface. In addition to the electrostatic attraction,hydrophobic or hydrophilic ligands are used to improve the bindingand/or release characteristics of the silica filter media by hydrophobicor hydrophilic interaction.

Treated silica filter media are characterized by measuring surface area,pore volume and pore size using methods known to the art such as aMicrometrics® analyzer. For example, surface area can be characterizedby BET technique. Pore volume and pore diameter can be calculated byBarrett-Joyner-Halenda analysis. Specific functional groups andmolecular structure can be determined by NMR spectroscopy.Carbon-hydrogen-nitrogen content can be determined by combustiontechniques; from this analysis information, the treatment level on theparticle surface can be calculated.

A sample, such as fermentation broth, can be applied to silane-treatedsilica filter media without pre-filtration. In one embodiment, thesample is mixed with the treated silica filter media by any means ofmechanical mixing (such as agitation, stirring, vortexing, etc.) for aperiod of time to allow sufficient binding of the component to thesurface of treated silica filter media. Those skilled in the art willrecognize that the time suitable for binding is dependent upon thecharacter of the pores of the media, the size of the target molecule orcomponent, the viscosity and other known mass transfer limitedprinciples. Generally, the time for binding to occur varies from about afew minutes to a few hours, but may continue up to 1-3 days. The mixtureis then filtered using a filtration unit. In another embodiment, asample can be filtered directly through a filtration unit containingsilane-treated silica filter media without pre-mixing the sample withthe filter media. The treated silica filter media capture particulatesand bind certain soluble components while allowing the unbound solublecomponents to flow through. The bound component is extracted by flowingan eluting solution through the filtration unit, and is recovered in aneluate stream. Useful eluting solutions include salt solutions, high pH(basic) solutions, low pH (acidic) solutions, chaotropic salts and otherreagents. Alternately, common organic solvents or mixtures thereof maybe used as long as they do not have deleterious affects on the recoveredcomponent of interest. Suitable high salts include NaCl, KCl, LiCl, etc.Suitable chaotropic salts include sodium perchlorate, guanidinehydrochloride, guanidine isothiocyanate, potassium iodide, etc. Otherreagents include urea and detergents. Combinations of the abovecomponents are also suitable in some applications. Alternately, aneluting solution is used to resuspend the surface silica filter media(containing particulates and bound molecules) by any means of mechanicalmixing for a period of time to allow sufficient release of the boundcomponent before filtering.

One application of the invention is to use the silane-treated silicafilter media to separate microorganisms from a desired product.Microbial contamination is a common problem across many industries,including brewing, winery, juice and beverages, dairy, industrial enzymeand pharmaceutical. Applicants have found that the silane-treated silicafilter media of this invention have anti-microbial activity. Bycontacting bacteria with the silane-treated silica filter media, thetotal viable bacterial counts are significantly reduced. Themicroorganisms are also captured by the silane-treated silica filtermedia. Thus, the filtration step further removes the microbialcontamination from the product.

The present invention is also directed to a silane-treated silica filtermedia having a general formula selected from the group consisting ofparticle-O—Si(R¹)_(x)(R²)_(3-x)R³,

wherein R¹ is alkoxy, halogen, hydroxyl, aryloxy, amino, carboxy, cyano,aminoacyl, or acylamino, alkyl ester, or aryl ester;

R² are independently substituted or unsubstituted alkyl, alkenyl,alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl,heterocyclic, cycloalkaryl, cycloakenylaryl, alkcycloalkaryl,alkcycloalkenyaryl, or arylalkaryl;

R³ is hydrogen, alkyl, alkenyl, alkaryl, alkcycloalkyl, aryl,cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, cycloalkaryl,cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, arylakaryl,alkoxy, halogen, hydroxyl, aryloxy, amino, alkyl ester, aryl ester,carboxy, sulphonate, cyano, aminoacyl, acylamino, epoxy, phosphonate,isothiouronium, thiouronium, alkylamino, quaternary ammonium,trialkylammonium, alkyl epoxy, alkyl urea, alkyl imidazole, oralkylisothiouronium; wherein the hydrogen of said alkyl, alkenyl, aryl,cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic is optionallysubstituted by halogen, hydroxyl, amino, carboxy, or cyano;

R⁵, R⁶ and R⁸ are independently hydrogen, substituted or unsubstitutedalkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl,heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl,alkcycloalkaryl, alkcycloalkenyaryl, or arylalkaryl;

R⁴, R⁷, R⁹ are substituted or unsubstituted alkyl, alkenyl, alkaryl,alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic,cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, orarylalkaryl radicals capable of forming two covalent attachments;

wherein said silica filter media is rice hull ash or oat hull ash.

The silane-reacted silica filter media of the present inventionpreferably have a functional moiety, which can react with a component ofinterest. The functional moiety is selected from the group consisting ofquaternary ammonium, epoxy, amino, urea, methacrylate, imidazole,sulphonate and other organic moieties known to react with biologicalmolecules.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting. Examples 1 through 5illustrate the surface treatment of silica filter media. Examples 6through 14 illustrate the use of the silane treated filter media forseparating one or more components of interest from a sample containingparticulate matter and soluble components. Examples 15-22 illustrate theantimicrobial activity of the silane-treated silica filter media and thefiltration results.

EXAMPLES Example 1 Preparation of Treated Rice Hull Ash Media (tRHA)Using Trialkoxysilanes in a Batch Process

The treatment equipment is composed of a 3-neck, round bottom reactionflask, a Teflon shaft mechanic stirrer, thermometer, condenser, andheating mantle around the flask. The reaction flask was loaded withungrounded RHA silica filter media (surface area: ˜30 m²/g), and solventmixture. Table 1 shows the reaction conditions for each example. Themixture was stirred for a few minutes at ambient temperature, then thesurface modification process involved addition of the proper amount ofthe silane directly to the mixture in a slow addition rate, while goodmixing was maintained. 250% of the proper amount of the silane ascalculated to achieve multilayer coverage (high-level treatment) or 85%of the amount of silane as calculated to achieve monolayer coverage (lowlevel treatment) was added and the silane quantity was also correctedfor their purity. The loading concentrations are also listed in Table 1.Subsequently, the mixture was heated to reflux under N₂ blanket, whichis used primarily for safety and has no other affect on the outcome ofthe reaction, although heating is not required. After 2 hours stirringand refluxing, the treated slurry mixture was allowed to cool. Then itwas transferred to a porcelain Büchner funnel outfitted with Whatmanfilter paper, and attached to a vacuum filter flash. The treated filterslurry was filtered and washed twice with toluene and twice with IPA.Afterward, the sample was dried in the hood for about 24 hours. Thetreated filter media was transferred to a Pyrex container and coveredwith a paraffin film having a number of holes made with a syringeneedle, and then the sample was dried in a vacuum oven at 60° C. for 2-4hours. The dried samples were analyzed for surface area, pore structure,and carbon-hydrogen-nitrogen content.

TABLE 1 Summary of treatment compositions and conditions Treatments aredone on low carbon, ungrounded RHA from Producers. Silica Silane AddedSample # Amount g Silane Type Treatment Condition Purity % Silane g 1 503-(trimethoxysilyl)propyloctadecyl- H₂O, reflux 42% 15.06dimethylammonium chloride 2 50 3-(trimethoxysilyl)propyloctadecyl- H₂O,room temp. 42% 15.06 dimethylammonium chloride 3 503-(trimethoxysilyl)propyloctadecyl- Toluene, reflux, 42% 15.06dimethylammonium chloride stochiometric H₂O 4 503-(trimethoxysilyl)propyloctadecyl- Toluene, IPA, reflux 42% 15.06dimethylammonium chloride 5 50 3-(trimethoxysilyl)propyloctadecyl-Toluene, IPA, reflux, 42% 15.06 dimethylammonium chloride stochiometricH₂O at end 6 50 3-(trimethoxysilyl)propyloctadecyl- Toluene, IPA, reflux42% 7.03 dimethylammonium chloride 7 503-(trimethoxysilyl)-2-(p,m-chloromethyl)- Toluene, IPA, reflux 90% 1.47phenylethane 8 50 3-(trimethoxysilyl)-2-(p,m-chloromethyl)- Toluene,IPA, reflux 90% 4.33 phenylethane 9 503-(N-styrylmethyl-2-aminoethylamino)- Toluene, IPA, reflux 32% 13.30propyltrimethoxysilane hydrochloride 10 503-(N-styrylmethyl-2-aminoethylamino)- Toluene, IPA, reflux 32% 4.99propyltrimethoxysilane hydrochloride 11 50N-trimethoxysilylpropyl-N,N,N- Toluene, IPA, reflux 50% 7.32trimethylammonium chloride 12 50 N-trimethoxysilylpropyl-N,N,N- Toluene,IPA, reflux 50% 2.49 trimethylammonium chloride 13 503-(N-styrylmethyl-2-aminoethylamino)- Toluene, IPA, reflux 40% 6.69propyltrimethoxysilane hydrochloride 14 503-(N-styrylmethyl-2-aminoethylamino)- Toluene, IPA, reflux 40% 19.67propyltrimethoxysilane hydrochloride 17 1003-aminopropyltrimethoxysilane Toluene, IPA, reflux 100% 7.52 18 1003-aminopropyltrimethoxysilane Toluene, IPA, reflux 100% 2.56 19 100N-(2-aminoethyl)-3- Toluene, IPA, reflux 97% 9.62aminopropyltrimethoxysilane 20 100 N-(2-aminoethyl)-3- Toluene, IPA,reflux 97% 3.27 aminopropyltrimethoxysilane 21 50Phenyldimethylethoxysilane Toluene, IPA, reflux 100% 1.82 22 50Phenyldimethylethoxysilane Toluene, IPA, reflux 100% 0.76 23 503-(trimethoxysilyl)propyl methacrylate Toluene, IPA, reflux 98% 7.66 2450 N-(triethoxysilylpropyl)urea Toluene, IPA, reflux 49% 5.44 25 50Trimethoxysilylpropyldiethylenetriamine Toluene, IPA, reflux 98% 2.73 2650 Bis(2-hydroxyethyl)-3- Toluene, IPA, reflux 58% 4.96aminopropyltriethoxysilane 27 50 N-[3-(triethoxysilyl)propyl]imidazoleToluene, IPA, reflux 96% 2.88

Example 2 Preparation of Different Types of Treated Silica Filter Media

Additional substrates, namely high carbon rice hull ash, different typesof ultra pure diatomaceous earth (Celpure P1000, Celpure P65), Celite545 (standard diatomaceous earth filter aid), Perlite, and LRA II (anon-silica based lipid adsorbent) were used. Table 2 summarizes thetreatment conditions and compositions of these samples.

TABLE 2 Compositions and conditions of treatments of differentsubstrates Loading Silica Substrate Silica Treatment Silane (X MonolayerAdded Sample # Media Type Amount g Treatment Type Condition Purity %coverage) Silane g 28 AgriSilicas 150 AEAPTMS Toluene, IPA, 97% 150%10.53 STD (A 0700) reflux 29 Celpure 100 AEAPTMS Toluene, IPA, 97% 180%0.51 P1000 (A 0700) reflux 30 Celpure 50 AEAPTMS Toluene, IPA, 97% 1070%1.53 P1000 (A 0700) reflux 31 Celpure 50 Z-6032 Toluene, IPA, 32% 200%1.46 P1000 (SMAEAPTMS) reflux 32 Perlite 50 AEAPTMS Toluene, IPA, 97%200% 0.24 (A 0700) reflux 33 Perlite 50 Z-6032 Toluene, IPA, 32% 200%1.21 (SMAEAPTMS) reflux 34 Celite 545 50 AEAPTMS Toluene, IPA, 97% 200%0.40 (A 0700) reflux 35 Celite 545 50 Z-6032 Toluene, IPA, 32% 200% 2.05(SMAEAPTMS) reflux 36 Celpure P65 50 AEAPTMS Toluene, IPA, 97% 200% 0.61(A 0700) reflux 37 Celpure P65 50 Z-6032 Toluene, IPA, 32% 200% 3.13(SMAEAPTMS) reflux 38 LRA II 50 AEAPTMS Toluene, IPA, 97% 120% 8.96 (A0700) reflux 39 LRA II 50 Z-6032 Toluene, IPA, 32% 120% 45.80(SMAEAPTMS) reflux Z-6032:3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilanehydrochloride AEAPTMS: N-(2-aminoethyl)-3-aminopropyltrimethoxysilane

Example 3 Two-step Process to Synthesize Hydrophilic Quaternary AmmoniumFunctional Filter Aids (Filter Media Samples 40 and 42)

The treatment equipment was composed of a 500 milliliter, 3-neck roundbottom reaction flask, a Teflon shaft mechanic stirrer, thermometer,condenser, and heating mantle around the flask. The reaction flask wasloaded with 50 g of amino-functional pretreated RHA (sample 17 or 19)silica filter media, and 200 ml IPA solvent. The mixture was stirred forfew minutes at ambient temperature, then the surface modificationprocess involved addition of the proper amount ofglycidyltrimethylammonium chloride (2.46 g for sample 17, or 2.02 g forsample 19) directly to the mixture in a slow addition rate, while goodmixing was maintained. The reaction mixture was heated and refluxedunder a N₂ blanket. After 4 hours stirring and refluxing, the treatedslurry mixture was allowed to cool. Then it was transferred to aporcelain Büchner funnel outfitted with Whatman filter paper, andattached to a vacuum filter flask. The treated filter cake was filteredand washed four times with about 150 ml of DI water each time.Afterward, the sample was dried in the hood for about 24 hours. Next thetreated silica filter media was transferred to a Pyrex container andcovered with a paraffin film having a number of holes made with asyringe needle, and then the sample was vacuum oven dried at 60° C. for2-4 hours. The dried samples were analyzed for surface area, poresstructure, carbon-hydrogen-nitrogen content, ²⁹Si NMR.

Example 4 Two-step Process to Synthesize HydrophilicSulphonate-Functional Filter Aids (Filter Media Sample 41)

The treatment equipment was composed of a 500 milliliter, 3-neck roundbottom reaction flask, a Teflon shaft mechanic stirrer, thermometer,condenser, and heating mantle around the flask. The reaction flask wasloaded with 50 g of epoxy-functional pretreated RHA silica filter media(sample 15), and 200 ml IPA:H₂O (5:1) solvent. The mixture was stirredfor few minutes at ambient temperature, and the reaction mixture heatedup to 70° C. under a N₂ blanket. The surface modification processinvolved addition of the mixture of 0.55 g of sodium metabisulfite, 0.07g of sodium sulfite catalyst, and 5 g water from an additional funneldirectly to the mixture in a slow addition rate over 1-2 hours, whilegood mixing was maintained. The temperature was then raised up toapproximately 80° C., until the reaction completed. The reaction wasmonitored by iodometric titration of residual NaHSO₃. Afterapproximately 22 hours stirring and refluxing, the treated slurrymixture was allowed to cool. Then it was transferred to a porcelainBüchner funnel outfitted with Whatman filter paper, and attached to avacuum filter flask. The treated filter cake was filtered and washedfour times with about 150 ml of DI water each time. Afterward, thesample was dried in the hood for about 24 hours. Next the treated filteraid was transferred to a Pyrex container and covered with a paraffinfilm having a number of holes made with a syringe needle, and then thesample was vacuum oven dried at 60° C. for 2-4 hours. The dried sampleswere analyzed for surface area, pores structure,carbon-hydrogen-nitrogen content, ²⁹Si NMR. Table 3 summarizescompositions and conditions of the two-step processes.

TABLE 3 Compositions and conditions of treatments of two step processes.Silica Silane Added Sample # Amount g 2nd Step Reactant TreatmentCondition Purity % Silane g 40 50 Glycidyltrimethylammonium chlorideIPA, reflux 75% 2.02 41 50 Na₂S₂O₅/Na₂SO₃ IPA, water, reflux 100%0.55/0.07 42 50 Glycidyltrimethylammonium chloride IPA, reflux 75% 2.46Characterization of the Treated Silica Filter Media: BET Surface Area,Pore Volume, Pore Diameter

The surface area and porosity were measured using a Micrometrics® ASAP2010 analyzer. Before analyses, the samples were degassed under vacuumat 150° C. until a constant pressure was achieved. In the analysis step,N₂ gas was adsorbed by the sample at 77° K. and the surface area wascalculated from the volume of adsorbate. BET parameters were acquired byintegration of the BET equation using ASAP-2010 software. Surface areawas calculated in the range of 0.05≦P/Po≦0.3 from the adsorption branchof the isotherm. Barrett-Joyner-Halenda analysis was used to calculatethe pore volume and pore diameter.

NMR

Identification of specific functional groups and molecular structure wasdetermined by ²⁹Si solid state NMR spectroscopy on a Unity Plus 400 MHzSpectrometer using a Varian VT CPMAS probe and a 7 mm motor.

Carbon-Hydrogen-Nitrogen (CHN)

CHN content was determined by combustion technique at Robertson MicrolitLaboratories. From this analysis information, the treatment level on thesurface was calculated.

Table 4 summarizes the characterization data of the treated silicasamples.

TABLE 4 Characterization data summary of treated silica samples % CSurface Pore by Moisture Area Volume Pore Robertson Ligand densitySample # content % m²/g cm³/g Diameter Å Microlit μmol/m² μmol/g 1 2.63%8.69 0.047 149.54 5.69% 23.73 206.16 2 4.43% 11.58 0.060 142.22 5.58%17.47 202.17 3 2.05% 17.85 0.077 98.42 5.12% 10.39 185.51 4 1.60% 22.510.097 97.05 3.11% 4.61 103.67 5 1.43% 23.45 0.098 93.15 2.96% 4.57107.25 6 1.89% 24.53 0.104 94.57 2.47% 3.36 82.33 7 1.57% 32.65 0.12899.68 0.84% 1.95 63.64 8 2.60% 33.66 0.129 99.64 1.01% 2.27 76.52 92.20% 22.98 0.101 105.56 2.19% 4.96 114.06 10 1.46% 29.32 0.118 96.801.32% 2.35 68.75 11 1.33% 30.24 0.124 100.45 1.67% 5.75 173.96 12 1.44%22.39 0.103 112.07 0.88% 4.09 91.67 13 1.59% 28.19 0.112 95.47 2.09%3.86 108.85 14 1.77% 18.76 0.077 101.39 2.98% 8.27 155.21 17 2.71% 28.020.100 97.28 1.36% 8.09 226.67 18 0.86% 30.48 0.118 100.00 0.72% 3.94120.00 19 1.49% 23.64 0.101 101.93 1.68% 8.46 200.00 20 1.75% 28.150.118 98.55 1.03% 4.36 122.62 21 1.44% 32.32 0.131 102.99 0.42% 1.3543.75 22 2.47% 32.28 0.133 104.50 0.23% 0.74 23.96 23 0.80% 29.80 0.12097.08 0.98% 3.04 90.74 24 1.05% 28.99 0.119 100.14 0.80% 2.87 83.33 252.06% 27.02 0.117 100.15 1.14% 3.91 105.56 26 0.96% 31.75 0.128 100.930.74% 1.77 56.06 27 1.63% 31.06 0.129 102.94 0.62% 1.66 51.67 28 2.90%16.11 0.023 215.71 0.82% 6.06 97.62 29 0.33% 2.18 0.002 106.61 0.09%4.92 10.71 31 0.04% 2.39 0.003 140.36 0.46% 10.02 23.96 33 5.68% 3.070.003 148.64 0.57% 9.66 29.69 34 0.48% 1.47 0.002 104.07 0.16% 12.9419.05 35 0.05% 2.11 0.002 139.39 0.22% 5.42 11.46 37 0.94% 5.66 0.014145.31 0.39% 3.59 20.31 39 5.31% 112.73 0.741 222.48 8.71% 4.02 453.6540 2.77% 21.82 0.099 105.43 1.82% 5.35 116.67 41 2.69% 29.02 0.114 98.120.99% 3.55 103.13 42 1.91% 26.17 0.109 102.99 1.41% 4.08 106.82

Example 5 Compositions and Treatment Conditions of Silica Filters andtheir Characterization

Table 5A-5C summarized additional compositions and treatment conditionsof rice hull ash and their characterization.

TABLE 5A Treatment Preparation Filter Reagent Me- gram Results diaGlycidyl- Ligand Sam- trimethyl- Density ple Treatment Silica SilicaFirst Additive Second Additive ammonium % C μmol/ No Type Type gram NameGram Name Gram chloride % m² 43 3-(N-styrylmethyl-2- Producers 253-(N-styrylmethyl-2- 19.83 5.61% 25.68 aminoethylamino)- RHAaminoethylamino)- propyltrimethoxy- propyltrimethoxy-silane silanehydrochloride hydrochloride 44 3-(Trimethoxysilyl Producers 253-(Trimethoxysilyl 3.88 1.06% 11.34 propyl) RHA propyl) isothiouroniumisothiouronium chloride chloride 45 3-(Trimethoxysilyl- Producers 25 3-3.88 1.67% 18.27 propyl) RHA (Trimethoxysilylpropyl) isothiouroniumisothiouronium chloride chloride 46 N-Octadecyldimethyl RiceSil 500 N-93.29 N- 4.12 2.46% 5.41 (3-Trimethoxysilyl 100 Octadecyldimethyl(3-(Triethoxysilyl- propyl) Trimethoxysilylpropyl) propyl)-o- ammoniumchloride, ammonium chloride polyethylene then N- oxide urethane(Triethoxysilyl- propyl)-o- polyethylene oxide urethane 473-(N-styrylmethyl-2- RiceSil 500 3-(N-styrylmethyl-2- 92.66 N- 3.121.96% 6.37 aminoethylamino)- 100 aminoethylamino)- (Triethoxysilyl-propyl propyltrimethoxysilane propyl)-o- trimethoxysilane hydrochloridepolyethylene hydrochloride, then oxide urethane N-(Triethoxysilylpropyl)- o-polyethylene oxide urethane 483-(N-styrylmethyl-2- RiceSil 500 3-(N-styrylmethyl-2- 185.33 4.16% 40.42aminoethylamino)- 100 aminoethylamino)- propyltrimethoxy-propyltrimethoxy-silane silane hydrochloride hydrochloride 49 3- RiceSil25 3-(Trimethoxysilyl- 3.88 1.90% 24.60 (Trimethoxysilylpropyl) 100propyl) isothiouronium isothiouronium chloride chloride 50N-(2-Aminoethyl)-3- RiceSil 500 N-(2-Aminoethyl)-3- 43.42 23.47 2.53%16.35 amino- 100 aminopropyltrimethoxy- propyltrimethoxysilane, silaneplus Glycidyltrimethyl- ammonium chloride 51 3- RiceSil 200 3- 21.531.00% 6.75 (Trihydroxysilylpropyl- 100 (Trihydroxysilylpropyl-methylphosphonate) methylphosphonate) sodium salt sodium salt 52 N-RiceSil 500 N- 9.33 0.68% 0.90 Octadecyldimethyl(3- 100Octadecyldimethyl(3- Trimethoxysilylpropyl) Trimethoxysilylpropyl)ammonium ammonium chloride chloride 53 N-(Trimethoxysilyl- RiceSil 500N- 5.80 1.50% 12.53 propyl) 100 (Trimethoxysilylpropyl) ethylenediamine,ethylenediamine, triacetic acid, triacetic acid, trisodium trisodiumsalt salt

TABLE 5B Silane Added Sample Silica Treatment Purity Silane g Ligand # gSilane Type condition % g % C Density 54 500trimethoxysilylpropyl-ethylenediamine, Toluene, Reflux, 45.0 72.46 1.864.79 triacetic acid, trisodium salt H₂O 55 500N-(triethoxysilylpropyl)-O- Toluene, Reflux, 95.0 59.68 2.34 4.39polyethylene oxide urethane H₂O 56 500 Bis-(2-hydroxyethyl)-3- Toluene,Reflux, 57.6 22.55 0.93 1.26 aminopropyltriethoxysilane H₂O 57 500((chloromethyl)phenylethyl)trimethoxy Toluene, Reflux, 90.0 8.70 1.051.55 silane H₂O 58 500 N-(3-triethoxysilylpropyl)-gluconamide Toluene,Reflux, 50.0 31.64 1.12 2.1 H₂O 59 500 3-mercaptopropyltriethoxysilaneToluene, Reflux, 95.0 9.95 0.81 3.59 H₂O 60 500N-(triethoxysilylpropyl)-4- Toluene, Reflux, 100.0 12.16 1.16 0.21hydroxybutyramide H₂O 61 500 3-(triethoxysilyl)propylsuccinic Toluene,Reflux, 95.0 12.73 0.76 1.46 anhydride H₂O 62 500 Tris(3- Toluene,Reflux, 95.0 34.18 1.28 2.13 trimethoxysilylpropyl)isocyanurate H₂O 63500 2-Hydroxy-4-(3-triethoxysilylpropoxy)- Toluene, Reflux, 95.0 23.231.61 1.74 diphenylketone H₂O 64 500 UreidopropyltrimethoxysilaneToluene, Reflux, 100.0 11.72 0.86 2.03 H₂O 65 5003-isocyanatopropyltriethoxysilane Toluene, Reflux, 95.0 6.90 0.81 5.31H₂O 66 500 N-(3-trimethoxysilylpropyl)pyrrole Toluene, Reflux, 100.06.08 0.87 3.26 H₂O 67 500 Bis[(3-methyldimethoxysilyl)propyl]- Toluene,Reflux, 100.0 18.92 1.72 1.4 polypropylene oxide H₂O

TABLE 5C Ricesil Ligand 100 Silane Silane % C Density Sample TreatmentWeight Purity Weight Robertson Calculated # Silane Type condition gram %gram Microlit umol/m2 68 trimethoxysilylpropyl- Toluene, 500 45% 24.201.08 1.27 ethylenediamine, triacetic acid, Reflux, H₂O trisodium salt 69N-trimethoxysilylpropyl-N,N,N—Cl, Toluene, 500 50% 14.60 0.79 1.28trimethylammonium chloride Reflux, H₂O 70 2-(4-chlorosulfonylphenyl)-Toluene, 500 50% 24.20 1.28 2.89 ethyltrichlorosilane Reflux, H₂O 713-(N-styrylmethyl-2- Toluene, 500 32% 46.30 1.65 4.69 aminoethylamino)-Reflux, H₂O propyltrimethoxysilane hydrochloride 72triethoxysilylpropylethyl-carbamate Toluene, 500 100% 12.35 1.01 1.60Reflux, H₂O 73 N-(triethoxysilylpropyl)-O- Toluene, 500 95% 19.94 1.091.01 polyethylene oxide urethane Reflux, H₂O 74 3- Toluene, 500 42%22.45 0.83 2.82 trihydrosilylpropylmethylphosphonate, Reflux, H₂O sodiumsalt 75 Bis-(2-hydroxyethyl)-3- Toluene, 500 58% 22.55 0.93 1.26aminopropyltriethoxysilane Reflux, H₂O 76N-(3-triethoxysilylpropyl)-4,5- Toluene, 500 96% 12.06 1 1.57dihydroimidazole Reflux, H₂O 77((chloromethyl)phenylethyl)trimethoxysilane Toluene, 500 90% 8.70 1.051.55 Reflux, H₂O 78 3-aminopropyltrimethoxysilane, Toluene, 500 81% 8.150.99 2.58 then Reflux, H₂O N-(triethoxysilylpropyl)-O- 95% 5.03polyethylene oxide urethane 79 3- Toluene, 500 42% 16.87 0.77 2.43trihydrosilylpropylmethylphosphonate, Reflux, H₂O sodium salt, thenN-(triethoxysilylpropyl)-O- 95% 5.02 polyethylene oxide urethane 80N-trimethoxysilylpropyl-N,N,N—Cl, Toluene, 500 50% 15.30 0.95 2.41trimethylammonium chloride, then Reflux, H₂O (3- 100% 2.40glycidoxypropyl)trimethoxysilane 81 3- Toluene, 500 42% 16.90 0.98 3.81trihydrosilylpropylmethylphosphonate, Reflux, H₂O sodium salt, thenBis-(2-hydroxyethyl)-3- 58% 5.31 aminopropyltriethoxysilane 823-(N-styrylmethyl-2- Toluene, 500 32% 34.76 1.72 4.95 aminoethylamino)-Reflux, H₂O propyltrimethoxysilane hydrochloride, thenN-(triethoxysilylpropyl)-O- 95% 5.04 polyethylene oxide urethane 832-(trimethoxysilylethyl)pyridine Toluene, 500 100% 9.01 1.17 2.89Reflux, H₂O 84 N-(3-triethoxysilylpropyl)- Toluene, 500 50% 31.64 1.122.10 gluconamide Reflux, H₂O 85 2-(trimethoxysilylethyl)pyridine,Toluene, 500 100% 6.74 1.04 2.40 then Reflux, H₂ON-(3-triethoxysilylpropyl)- 50% 7.90 gluconamide 863-mercaptopropyltriethoxysilane Toluene, 500 95% 9.95 0.81 3.59 Reflux,H₂O 87 N-trimethoxysilylpropyl-N,N,N—Cl, Toluene, 500 50% 15.30 0.982.54 trimethylammonium chloride, then Reflux, H₂ON-(3-triethoxysilylpropyl)- 50% 7.95 gluconamide 88N-(triethoxysilylpropyl)-4- Toluene, 500 100% 12.16 1.16 0.21hydroxybutyramide Reflux, H₂O 89 3-(triethoxysilyl)propylsuccinicToluene, 500 95% 12.73 0.78 1.42 anhydride Reflux, H₂O 90Trimethoxysilylpropyl Toluene, 50 50% 1.00 1.04 1.43 polyethyleneimineReflux, H₂O 91 Tris(3- Toluene, 500 95% 34.18 1.28 1.60trimethoxysilylpropyl)isocyanurate Reflux, H₂O 92 2-Hydroxy-4-(3-Toluene, 500 95% 23.23 1.61 1.74 triethoxysilylpropoxy)- Reflux, H₂Odiphenylketone 93 Ureidopropyltrimethoxysilane Toluene, 500 100% 11.720.86 2.03 Reflux, H₂O 94 O-(propargyloxy)-N- Toluene, 500 90% 17.77 1.041.84 (triethoxysilylpropyl)urethane Reflux, H₂O 95 3- Toluene, 500 42%9.33 0.21 0.24 (trimethoxysilyl)propyloctadecyldi Reflux, H₂Omethylammonium chloride 96 N-1-phenylethyl-N′- Toluene, 500 100% 9.701.02 2.43 triethoxysilylpropylurea Reflux, H₂O 973-isocyanatopropyltriethoxysilane Toluene, 500 95% 6.90 0.81 5.31Reflux, H₂O 98 2-(3,4- Toluene, 500 97% 6.90 0.98 3.22epoxycyclohexyl)ethyltrimethoxysilane Reflux, H₂O 99N-(3-trimethoxysilylpropyl)pyrrole Toluene, 500 100% 6.08 0.87 3.26Reflux, H₂O 100 Bis[(3- Toluene, 500 100% 18.96 1.07 1.40methyldimethoxysilyl)propyl]- Reflux, H₂O polypropylene oxide 101N-trimethoxysilylpropyl-N,N,N—Cl, Toluene, 500 50% 10.34 0.84 3.67trimethylammonium chloride, then Reflux, H₂O 2-Hydroxy-4-(3- 95% 3.12triethoxysilylpropoxy)- diphenylketone 102Trimethoxysilylpropylisothiouronium Toluene, 500 43% 15.55 0.71 0.44chloride Reflux, H₂O 103 (3- Toluene, 500 100% 6.23 0.74 0.35glycidoxypropyl)trimethoxysilane Reflux, H₂O 1043-mercaptopropyltriethoxysilane, Toluene, 500 95% 4.98 0.79 1.14 thenReflux, H₂O N-(triethoxysilylpropyl)-O- 95% 3.38 polyethylene oxideurethane 105 3-(triethoxysilyl)propylsuccinic Toluene, 500 95% 6.36 0.941.05 anhydride, then Reflux, H₂O N-(triethoxysilylpropyl)-O- 95% 3.40polyethylene oxide urethane 106 trimethoxysilylpropyl- Toluene, 500 45%20.35 1.16 1.64 ethylenediamine, triacetic acid, Reflux, H₂O trisodiumsalt, then N-(triethoxysilylpropyl)-O- 95% 3.45 polyethylene oxideurethane 107 2-(4-chlorosulfonylphenyl)- Toluene, 500 50% 13.40 0.930.89 ethyltrichlorosilane, then Reflux, H₂O N-(triethoxysilylpropyl)-O-95% 3.40 polyethylene oxide urethane 108 2-(4-chlorosulfonylphenyl)-Toluene, 500 50% 15.30 1.01 1.15 ethyltrichlorosilane, then Reflux, H₂OBis-(2-hydroxyethyl)-3- 58% 5.32 aminopropyltriethoxysilane Liganddensities are corrected for 0.43% C due to residual carbon on theoriginal rice hull ash. Mixed silanes sample ligand density are based onfirst silane.

Example 6 Surface Treated Rice Hull Ash for Protein Binding and ReleaseObjective

To test the binding and release of protein using surface treated ricehull ash (RHA). The protein solution is particulate free, derived fromMicrococcus luteus fermentation.

Table 6 summarizes the filter media samples and their surfacetreatments.

TABLE 6 Sample Designation Treatment 63-(trimethoxysilyl)propyloctadecyl- dimethylammonium chloride treatedRHA 4 3-(trimethoxysilyl)propyloctadecyl- dimethylammonium chloridetreated RHA Unground RHA Untreated from Producers FW12 Commercialdiatomaceous earth (Eager Picher) HQ50 Commercial quaternary amineion-exchange resin (PerSeptive BioSystems)Procedure

-   -   1.2 g of each sample was measured into a 50-mL conical tube.    -   2. 25 mL of 25 mM Tris-HCL, pH 8.4 buffer was added.    -   3. Sample and buffer were mixed by inversion overnight.    -   4. Each of the wetted samples was transferred to a 15 mL conical        tube, then centrifuged at 2500 g for 5 minutes and the        supernatant was decanted. The resulting samples were used for        binding test below.    -   5. Protein test solution description and preparation:        -   Source: Micrococcus luteus particulate free concentrated            broth recovered using the following steps:            -   Fermentation broth was lysed using 200 ppm lysozyme                (from chicken hen white).            -   Lysed broth was flocculated using a poly-cationic                polymer and filtered to remove particulates.            -   Particulate broth was concentrated using an ultrafilter                to dewater (Prep/Scale™ TFF, Millipore).    -   6. The above solution was adjusted with 24 parts of 25 mM        Tris-HCl pH 8.4 buffer.    -   7. 5 mL of protein test solution was added to each tube        containing surface treated rice hull ash.    -   8. The tubes were mixed by inversion for 90 min.    -   9. The mixed tubes were centrifuged at 2500 g for 5 minutes and        the supernatant was decanted. The fraction collected is referred        to as “Flow Through or FT”.    -   10. 5 mL of 25 mM Tris-HCl pH 8.4 buffer was added to each of        the tubes which were allowed to mix by inversion for 45 min.    -   11. The tubes were centrifuged at 2500 g for 5 min and the        supernatant was decanted. The fraction collected is referred to        as “Wash”.    -   12. 5 mL elution buffer (25 mM Tris-HCl pH 8.4 containing 2M        NaCl) was added and mixed for 30 min.    -   13. 0.5 mL of 0.5M NaOH was added to each tube.    -   14. The tubes were mixed by inversion for 90 min.    -   15. The tubes were centrifuged at 2500 g for 5 min and the        supernatant was decanted. The fraction collected is referred to        as “Eluate”.    -   16. All the fractions were analyzed by SDS-PAGE gel        electrophoresis (procedure according to NuPAGE Electrophoresis        System, U.S. Pat. No. 5,578,180, by NOVEX electrophoresis GmbH,        Germany).        Observations        FIG. 1A (Binding and Analysis of Unbound Components)        Unbound sample was detected by analysis of the Flow Through and        the Eluate represents all or a portion of bound sample released        in the elution process.    -   FW12 (commercial diatomaceous earth) did not bind any protein        from the feed (lane #3 versus lane #2). The slightly lower        intensity for all the bands is due to the dilution by the        solution used to pre-wet the test sample.    -   Untreated RHA selectively bound a protein band above 6 kd and        below 14.4 kd (lane #4 versus lane #2). The slightly lower        intensity for all the bands is due to the dilution by the        solution used to pre-wet the test sample.    -   HQ50 (commercial quaternary amine ion-exchange resin) bound most        of the proteins from the test solution except below 14.4 kds        (lane #5 versus lane #2)    -   Treated RHA Sample 4 selectively bound near and above 97 kd        region, between 55.4 and 36.5 kd, near 21 kd and 14.4 kd        proteins. Note that the bands below 14.4 kd were not captured,        as in the case with HQ50. The overall protein band intensity        appears lower than the untreated rice hull ash and FW12, which        suggests greater binding by treated RHA.    -   Treated RHA Sample 6 demonstrated similar protein binding        selectivity as sample 4 but appears to have lower binding        capacity. Note that the bands below 14.4 kd were not captured,        as in the case with HQ50. The overall protein band intensity        appears lower than the untreated rice hull ash and FW12.        FIG. 1B (Release and Analysis of Bound Components)    -   FW12 eluate contains trace amount of proteins which are most        likely from the physically trapped/carried over liquid (lane        #2).    -   The protein, below 14.4 and above 6 kd bands, bound to untreated        RHA were released (lane #3)    -   All the proteins captured by HQ50 were released (lane #4)    -   Eluate from sample 4 contains protein bands above 116 kd, near        and below 55 Kd and near 6 kd. The above 36.5 kd band appears to        remain bound (lane #5).    -   Eluate from sample 6 contain mostly above 116 kd and near 55 kd        bands. Others that were bound either remain bound or are too low        to be detected by the analysis (lane #6).        Untreated diatomaceous earth did not exhibit protein-binding        capability. The untreated rice hull ash demonstrated some        protein binding capability. The two treated rice hull ashes,        sample 4 and 6, demonstrate protein-binding capability.

Example 7 Surface Treated Rice Hull Ash for Protein Binding and Release

Objective

To test the binding and release of protein using additional surfacetreated rice hull ash. The protein solution is particulate free, derivedfrom Micrococcus luteus fermentation.

Table 7 summarizes the filter media samples and their surfacetreatments.

TABLE 7 Sample Designation Treatment 73-(trimethoxysilyl)-2-(p,m-chloromethyl)-phenylethane treated 83-(trimethoxysilyl)-2-(p,m-chloromethyl)-phenylethane treated 93-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilanehydrochloride treated 10 3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride treated 11N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride treated 12N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride treatedProcedureSame as in Example 6.ObservationsSample 7 Protein Binding and Release (FIG. 2A)

-   -   Selectively bound all MW bands below 55 kd except near 21.5 kd        (lane 2 versus lane 3).    -   The wash has similar profile compared to flow through.    -   The eluate has a very light band near 55 kd, and not many other        bands. The other bands appear to be tightly bound and were not        eluted under conditions used.        Sample 8 Protein Binding and Release (FIG. 2A)    -   Similar observations as above, Sample 7.        Sample 9 Protein Binding and Release (FIG. 2B)    -   Almost all the proteins were bound except for the band below 14        kd (lane 1 versus lane 2)    -   No protein bands were detected in the wash fraction.    -   Eluate fraction contained mostly near 55 kd band, other bands        remain bound. This demonstrated selective release for the near        55 kd band resulting in a protein purity >90+% based on band        intensity.        Sample 10 Protein Binding and Release (FIG. 2B)    -   Similar observations as the sample 9 above        Sample 11 Protein Binding and Release (FIG. 2C)    -   Almost all, except some low MW bands, were bound. This        demonstrated selective protein binding. (lane 1 versus lane 3)    -   No protein bands were detected in the wash fraction.    -   Most of the bands bound were eluted under conditions used (lane        5).    -   Appears to have relatively high binding capacity compared to        other surface treated rice hull ashes.        Sample 12 Protein Binding and Release (FIG. 2C)        Similar Observations as the Sample 9 Above        Conclusions        Unique protein binding and release were observed for surface        treated rice hull ashes. Selective binding was observed (Sample        7 and Sample 8). Selective release (sample 9 and sample 10)        resulted in >90% protein purity fractions.

Example 8 Surface Treated Rice Hull Ash for Protein Binding and ReleaseObjective

To test the binding and release of protein using additional surfacetreated rice hull ash. The experiment design is based on ion exchange.The protein solution is particulate free, derived from Micrococcusluteus fermentation.

Table 8 summarizes the filter media samples and their surfacetreatments.

TABLE 8 Treated Rice Hull Ash Identification Surface Treatment 143-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilanehydrochloride treated 13 3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride treated 173-aminopropyltrimethoxysilane treated 18 3-aminopropyltrimethoxysilanetreated 19 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated 20N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated surfaceProcedure

-   1.2 g of each surface treated rice hull ash was weighed into a 50 mL    conical tube and 40 mL equilibration buffer (25 mM Tris-HCl pH 8.4)    was added. The tubes were mixed by inversion for 30 min.-   2. The tubes were centrifuged at 2500×g for 5 minutes and the    supernatant was decanted.-   3. Protein test solution source: Micrococcus luteus particulate free    concentrated broth was prepared as in Example I followed by partial    digestion using 10 ppm protease.-   4. The above solution was adjusted with 24 parts of 25 mM Tris-HCl    pH 8.4 buffer.-   5. 20 mL of protein test solution was added to each prepared surface    treated rice hull ash.-   6. The samples were mixed by inversion for 30 min.-   7. The samples were centrifuged at 2500×g for 5 minutes and the    supernatant was decanted. The fraction collected is referred to as    “Flow Through or FT”-   8. 20 mL of 25 mM Tris-HCl pH 8.4 buffer was added to the samples    which were allowed to mix by inversion for 15 min.-   9. The samples were centrifuged at 2500×g for 5 min and the    supernatant was decanted. The fraction collected is referred to as    “Wash”.-   10. 20 mL elution buffer (25 mM Tris-HCl pH 8.4 containing 1M NaCl)    was added to each sample and the samples were mixed by inversion for    30 min.-   11. The samples were centrifuged at 2500×g for 5 min and the    supernatant was decanted. The fraction collected is referred to as    “Eluate #1”-   12. Steps 9 and 10 were repeated using 10 mL of the same elution    buffer+50 mM NaOH. The fraction collected is referred to as “Eluate    #2”.-   13. All of the fractions were analyzed by SDS-PAGE gel    electrophoresis.    Observations    Sample 14 Protein Binding and Release (FIG. 3A)    -   The flow through fraction has relatively low protein band        intensity, which indicates sample 14 has relatively good binding        capacity (lane #6 versus lane #1)    -   The band below 14.4 kd remains in the flow through, which        indicates selective binding.    -   The bound proteins were partially eluted by 1M NaCl.    -   Addition of NaOH to the 1M NaCl containing buffer further eluted        the bound proteins.        Sample 13 Protein Binding and Release (FIG. 3B)    -   All the feed proteins were bound (lane #3 versus lane #2)    -   Only a small amount of bound protein was eluted at 1M NaCl (lane        #5), which suggests that the binding may not be ion exchange.    -   Addition of caustic to the 1M NaCl elution buffer successfully        eluted bound protein.    -   The behavior was similar to sample 14.        Sample 17 Protein Binding and Release (FIG. 3C)    -   Relatively good binding as shown in lane #7 flow through        fraction.    -   Selectively did not bind the below 14 kd protein.    -   Required high NaCl/NaOH for elution.        Sample 18 Protein Binding and Release (FIG. 3C)    -   Relatively good binding as shown in lane #1 flow through        fraction.    -   Selectively did not bind the below 14 kd protein.    -   Required high NaCl/NaOH for elution.    -   The results are similar to those of sample 17.        Sample 19 Protein Binding and Release (FIG. 3D)    -   Relatively good binding, as shown in the flow through fraction        on lane #7 having low protein bands.    -   Selectively did not bind the below 14 kd band.    -   Most of the bound proteins were eluted at 1M NaCl.    -   The addition of NaOH to 1M NaCl containing buffer further eluted        the near 55 kd bands.        Sample 20 Protein Binding and Release (FIG. 3E)    -   Relatively good binding as indicated by the low protein bands in        the flow through fraction (lane #3).    -   Some leakage during wash (lane #4).    -   Selectively did not bind the below 14 kd band (lane #3).    -   The bound proteins were eluted mostly at 1M NaCl (lane #5).    -   The results are similar to those of sample 19.        Conclusions        For the above surface treated rice hull ash samples tested,        three general binding/release behaviors were observed when the        samples were tested under conditions suitable for binding based        on anion exchange and release by high salt and/or high pH:        Relatively good binding, elute with NaCl/NaOH:        Sample 14        (3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane        hydrochloride treated)        Sample 13        (3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane        hydrochloride treated)        Sample 17 (3-aminopropyltrimethoxysilane treated)        Sample 18 (3-aminopropyltrimethoxysilane treated)        Relatively good binding, elute with NaCl:        Sample 19 (N-(2-aminoethyl)-3-aminopropyltrimethoxysilane        treated)        Sample 20 (N-(2-aminoethyl)-3-aminopropyltrimethoxysilane        treated)        The binding/release test was designed to test for anion exchange        behavior. The observations are consistent with the RHA surface        modifications.        The responses of sample 14 and sample 13 are consistent with a        combination of ion exchange and hydrophobic characteristics.        Sample 17 and sample 18 also demonstrated a mixture of        behaviors.        Sample 19 and sample 20 have typical characteristics similar to        anion-exchange behavior in terms of both binding and release.

Example 9 Surface Treated Rice Hull Ash for Protein Binding and Release(Cation Exchange)

Objective

To test the binding and release of protein using surface treated ricehull ash. The protein solution is particulate free, derived fromAspergillus niger fermentation.

Table 9 summarizes the samples designation and their surface treatments.

TABLE 9 Treated Rice Hull Ash Identification Surface 41 1^(st) step3-glycidoxypropyltrimethoxysilane and 2^(nd) step Na₂S₂O₅ treatmentUnground Untreated RHA from Producers Porous HS50 Commercial - SH cationexchange resin (PerSeptive BioSystems, Farmington, MA)Procedure

-   1.2 g of each surface treated rice hull ash were placed into a 50 mL    conical tube and 40 mL equilibration buffer (100 mM Sodium Acetate,    pH 4.0) was added. The tubes were mixed by inversion for 30 min.-   2. The tubes were centrifuged at 2500×g for 5 minutes and the    supernatant was decanted.-   3. Protein test solution description and preparation:    -   a. Source: Aspergillus niger particulate free concentrated broth        recovered using the following steps:        -   i. The fermentation broth was filtered to remove cell.        -   ii. Ultrafilter (dewater (Prep/Scale™ TFF, Millipore) cell            free broth to dewater.    -   b. The above solution was adjusted with 14 parts of 100 mM        Sodium Acetate, pH 4.0 buffer.-   4. 20 mL of protein test solution was added to each prepared surface    treated rice hull ash.-   5. The samples were mixed by inversion for 70 min.-   6. The samples were centrifuged at 2500×g for 5 minutes and the    supernatant was decanted. The fraction collected is referred to as    “Flow Through or FT”.-   7. 20 mL of 100 mM Sodium Acetate pH 4.0 buffer was added to each    sample, and the samples were allowed to mix by inversion for 15 min.-   8. The samples were centrifuged at 2500×g for 5 min and the    supernatant was decanted. The fraction collected is referred to as    “Wash #1”.-   9. Steps 7 & 8 were repeated and the fraction collected is referred    to as “Wash #2”-   10. 20 mL elution buffer (100 mM Sodium Acetate pH 4.0 buffer    containing 1M NaCl) was added and the samples were mixed by    inversion for 60 min.-   11. The samples were centrifuged at 2500 g for 5 min and the    supernatant was decanted. The fraction collected is referred to as    “Eluate #1”-   12. Repeated step 9 and 10 using 10 mL of the same elution buffer+50    mM NaOH. The fraction collected is referred to as “Eluate #2”.-   13. All the fractions were analyzed by SDS-PAGE gel electrophoresis.    Observations    Sample 41 Protein Binding and Release (FIG. 4A)    -   Selectively binds near 97 kd and below 31 kd bands.    -   There were relatively low to no protein bands detected in the        “Washes #1 and #2”, respectively (see lane #3 and lane #4,        respectively), which implies that the binding was        specific/strong.    -   The bound proteins were eluted in 1M NaCl containing buffer.        Untreated RHA Protein Binding and Release (FIG. 3A)    -   The near 97 kd and below 31 kd bands were not present in the        flow through. However, no proteins were eluted in either Eluate        #1 or Eluate #2.        Porous HS50: Protein Binding and Release (FIG. 4B)    -   Selectively binds near 97 kd and below 31 kd bands.    -   There were relatively low to no protein bands detected in the        “Washes #1 and #2”, respectively (see lane #3 and lane #4,        respectively), which suggests that the binding was        specific/strong.    -   The bound proteins were eluted in 1M NaCl containing buffer.        Conclusion        The surface treated rice hull ash sample 41 has very similar        binding and release characteristics to the positive control.

Example 10 Surface Treated Silica Filter Media for Protein Binding andRelease (Ion Exchange)

Objective

To test the binding and release of protein using surface treated silicafilter media. The experiment design is based on ion exchange. Theprotein solution is particulate free, derived from Micrococcus luteusfermentation.

Table 10 summarizes the samples designation and their surfacetreatments.

TABLE 10 Sample Identification Description Sample 42 1^(st) step3-aminopropyltrimethoxysilane and 2^(nd) step Glycidyltrimethylammoniumchloride treatment Sample 40 1^(st) stepN-(2-aminoethyl)-3-aminopropyltrimethoxysilane and 2^(nd) stepGlycidyltrimethylammonium chloride treatment Sample 34N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated Celite 545 Sample29 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated Celpure P1000(commercial diatomaceous earth) AgriSilicas Untreated RHA Celite 512Untreated commercial diatomaceous earth (World Minerals)ProcedureSame as in Example 8 for all samples except sample 29, sample 30 andCelPure P100, which have the following variations:The protein test solution was diluted by 10× (versus 25×).Steps 4 and 5 were repeated and the wash fraction collected is referredto as “Wash #2”.ObservationsUnder the test conditions used, the amount of protein test solution wasin excess. As a result, all the flow through fractions had similarprotein band patterns compared to the feed test solution. No attempt wasmade to qualitatively describe the protein binding capability of eachsilica filter media sample tested. The following observations are basedon the eluate fractions only.Sample 42 (FIG. 5A)Most of the bound proteins were eluted at 1M NaCl (lane #5).Sample 40 (FIG. 5B)Most of the bound proteins were eluted at 1M NaCl (lane #5).Sample 34 (FIG. 5C)No significant amount of protein was eluted at 1M NaCl.Small amount of proteins were eluted subsequently using high pH.Sample 29 (FIG. 5D)Both eluate fractions contain proteins, and the compositions seemsimilar in these fractions (lane #5 for 1M NaCl eluate and lane #6 forhigh pH eluate).Untreated AgriSilica RHA (FIG. 5B)The eluted fractions contain proteins, especially at MW lower than 14.4kd.Celite 512 (FIG. 5C)The fraction eluted at 1M contains proteins near 97 kd, near and below55 kd and especially between 14.4 kd and 6 kd (lane #10).ConclusionSamples 40 and 42 (surface treated rice hull ash) and samples 29 and 34(surface treated diatomaceous) demonstrate protein-binding capabilityover the corresponding untreated counterparts.

Example 11 Surface Treated Rice Hull Ash for Dynamic Protein Binding andRelease (Ion Exchange)

Objective

To test the dynamic binding and release of protein using surface treatedrice hull ash sample 9. The experiment design is based on ion exchange.The protein solution is particulate free, derived from Micrococcusluteus fermentation.

Procedure

-   1. 6 g of sample 9 was placed into a 50 mL conical tube.-   2. 50 mL equilibration buffer (25 mM Tris-HCl pH 8.4) was added and    the sample was mixed by inversion for 30 min.-   3. The samples was centrifuged at 2500 g for 5 minutes and the    supernatant was decanted.-   4. 30 mL of equilibration buffer was added and the sample was mixed    well by inversions.-   5. The sample was poured into a gravity flow column.-   6. The surface-treated rice hull ash was allowed to settle and pack    to a 10 mL volume.-   7. The pre-filter was placed onto the packed bed.-   8. 20 ml of equilibration buffer was added.-   9. 25 mL of protein test solution was added (prepared the same way    as in Example 6)-   10. Flow through fractions were collected in 15 mL conical tubes.-   11. 30 mL of equilibration buffer was added, and the “wash” was    collected in 15 ml conical tubes.-   12. The following steps were used sequentially for election and    collection of multiple elutes as shown in Table 11:    -   a. 0.2M NaCl in equilibration buffer was added.    -   b. 2M NaCl in equilibration buffer was added.    -   c. 0.1M NaOH was added.-   13. All the fractions were analyzed by SDS-PAGE gel electrophoresis.    Observations (FIG. 6)    -   The amount of solution loaded was higher than the capacity,        hence significant breakthrough in the FT fractions (lanes 3, 4        and 5)    -   The surface treated rice hull ash, sample 9, had good flow        property. All the steps performed above were easily accomplished        by gravity flow.    -   FIG. 6 shows that at the at 10 mL load, the feed solution        appears to breakthrough the 10 mL packed sample 9.    -   Under the binding conditions tested, sample 9 appears to        selectively bind the near 96 kd, near 55 kd, the two bands below        the 55 kd, bands near and between the 14.4 kd and 6 kd.    -   The following were observed with the three elution steps:        -   Three bands (near 97 kd, near 55 kd, and below 14.4 kd) were            eluted at 0.2M.        -   At 2M NaCl, near 97 kd and near 55 kd bands were eluted.        -   Under 0.1M NaOH, near 55 kd, below 31 kd bands and near 14.4            kd proteins were eluted.            Table 11 shows a summary of fractions collected for the            binding test.

TABLE 11 Volume Fraction (mL) Feed 25 mL loaded FT#1 10.5 mL FT#2 (after10 mL feed was loaded) 10 mL FT#3 (after 14.5 mL was loaded) 4.5 mL FT#4(after 25 mL feed was loaded) 10 mL Wash 30 mL Eluate #1 (0.2M NaCl) 10mL Eluate #2 (2M NaCl) 10 mL Eluate #3 (1st 0.1M NaOH fraction, verydark) 4.5 mL Eluate #4 (2^(nd) 0.1M NaOH) 3.5 mLConclusionsThis example demonstrates that surface-treated rice hull ash can be usedin a packed bed chromatography mode for protein binding and release andas a filter aid with gravity flow alone. The binding and releasecharacteristics are similar to those of batch mode. The example alsoillustrates that selective elution can be achieved by using differentelution buffers.

Example 12 Surface-Treated Rice Hull Ash for Simultaneous ParticulateCapture and Soluble Capture/Release

Objective

To test the characteristics of surface-treated rice hull ash forsimultaneous particulate filtration, and protein binding and release.The surface treated rice hull ash was designated sample 19, which wasdemonstrated to have anion exchange characteristics (see Example 8). Theuntreated rice hull ash was also tested in parallel.BuffersEquilibration Buffer: 25 mM Tris-HCl, pH 8.4.Elution Buffer: 25 mM Tris-HCl, 1M NaCl, pH 8.4; 1M NaOH; 1M HClTest Solution

Flocculated Micrococcus luteus fermentation broth referred to as “feed”was prepared according to the following:

-   -   After harvest, the broth was lyzed using 100 ppm lysozyme        (chicken egg white).    -   The lysed broth was flocculated using poly-cationic polymer.    -   The flocculated sample was diluted with 1 part equilibration        buffer before testing.        Procedure

-   1. Surface-treated rice hull ash preparation:    -   5 g of untreated RHA was placed into each of the two 50 mL        conical tubes.    -   40 mL equilibration buffer was added and the tubes were mixed by        inversion for 30 min.

-   2. The tubes were centrifuged at 2500×g for 5 minutes and decanted    in step #1 for the untreated rice hull ash.

-   3. 50 mL of the prepared test solution “feed” was added to each    prepared rice hull ash.

-   4. The tubes were mixed by inversion for 30 min at room temperature.

-   5. A 1 mL small sample was centrifuged using a bench top centrifuge    (4 min) and the supernatant was collected (referred to as “Bench    FT”).

-   6. 0.45 μm 250 mL-Nalgen unit was prepared for filtration:    -   The unit was connected to a house vacuum outlet.    -   The other prepared rice hull ash was suspended in 50 mL of        equilibration buffer.    -   The suspension was poured into the filter unit, and (house)        vacuum was applied to form a pre-coat (cake).    -   The filtrate reservoir was emptied.    -   The reservoir was reconnected for the filtration test.

-   7. The protein solution with rice hull ash ad-mix from step 4 was    poured into the prepared filtration unit and vacuum was reapplied to    start filtration. The collected filtrate sample is referred to as    “FT Filtrate”.

-   8. The vacuum was discontinued and 50 mL of Equilibration Buffer was    added and mixed by stirring. The vacuum was reapplied to start    filtration. The filtrate sample was collected and referred to as    “Wash”.

-   9. Step 8 was repeated with 50 mL of Elution Buffer and mixed for 15    min before vacuum was reapplied to start filtration. The filtrate    sample was collected and is referred to as “Eluate”.

-   10. All the fractions were analyzed by 4-12% Tris-Bis SDS-PAGE gel    electrophoresis with MES running buffer (see separate Excel file for    procedure).

-   11. Steps 1-10 were repeated with the untreated rice hull ash.    Observations/Comments

-   1. The surface-treated rice hull ash, sample 19, appears to have    slightly thinner cake thickness than the untreated rice hull ash.

-   2. All the fractions collected (FT filtrate, wash and eluate) from    both rice hull ashes were clear, free of particulate.

-   3. The surface-treated rice hull ash, sample 19, has a particulate    filtration rate comparable to the filtration rate of the untreated    rice hull ash:    -   Sample 19: 12.8 mL/min    -   Untreated RHA: 14.0 mL/min

-   4. Sample 19 demonstrates good capture and release over untreated    RHA:    -   Untreated RHA (FIG. 7A)        -   The “FT filtrate” (lane #4) has very similar profile as the            feed (lane #2). All the bands are slightly lighter than the            feed, which is an artifact of dilution from the buffer used            to condition the rice hull ash.        -   The protein solution physically trapped within the rice hull            ash was displaced and this is represented by the “wash”            fraction (contains very faint protein bands, see lane #5)        -   There was only trace amount of protein in the Eluate (lane            #6).

-   5. Sample 19 (FIG. 7B)    -   Demonstrates good binding and recovery of the bound protein.    -   The “FT filtrate” fraction has very low to no protein (lane #4).        The “Bench FT” supernatant (lane #3) has slight protein bands        when compare to the “FT Filtrate”, which indicates that proteins        were captured as they passed through the cake.    -   The wash has low to no protein bands (lane #5).    -   The Eluate has similar band patterns but slightly less intense        than the feed (lane #6).        Conclusion        The surface-treated rice hull ash simultaneously captured        soluble proteins of interest by ion exchange and separated        particulates from the feed protein solution. The captured        proteins can be subsequently extracted from the surface treated        rice hull ash by elution with a high-salt buffer.        The results demonstrate that surface-treated rice hull ash can        be used to separate a particulate-containing protein solution        into three streams:        particulates trapped in surface-treated rice hull ash pre-coat        and body feed,        non-protein components bound to surface treated rice hull ash,        and        protein components bound to and eluted off the surface treated        rice hull ash.

Example 13 Surface Treated Rice Hull Ash for Simultaneous ParticulateCapture and Soluble Capture/Release

Objective

To repeat Example 12 using a Aspergillus niger broth using the samesurface-treated rice hull ash (sample 19) and untreated rice hull ash.

Test Solution

Aspergillus niger fermentation was diluted with 4 parts of DI water andpH was adjusted to 8.06 using NaOH.

Procedure

Same as in the Example 12. Test solution volume was 100 mL.

Observations/Comments

The surface treated rice hull ash, sample 19, has a comparableparticulate filtration rate to the untreated rice hull ash.

All the fractions collected (FT filtrate, wash and eluate) from bothrice hull ashes were clear, free of particulate.

Under the conditions tested, the amount of test solution used was inexcess of the binding capacity. As a result, the flow through fractions(both “bench FT” and “Filtrate FT”) for both sample 19 and untreated RHAwere not significantly different from the feed solution. See FIG. 8,lanes #2, 3 and 4 versus lane #1 for untreated RHA and lanes #7, 8 and 9versus lane #1 for sample 19.The following observations confirmed that sample 19 has protein-bindingcapability over the untreated RHA (see FIG. 8):Untreated RHA Wash (lane #5) contains more protein than the sample 19(lane #10).The eluted fraction from sample 19 (lane #11) shows higher protein bandintensity than the eluted fraction from untreated RHA (lane #6).ConclusionThis example demonstrates that the surface-treated rice hull ashsimultaneously captured soluble proteins of interest by ion exchange andseparated particulates from the Aspergillus niger derived feed proteinsolution. The captured proteins can be subsequently extracted from thesurface treated rice hull ash by elution with high salt buffer.The results demonstrate that surface-treated rice hull ash can be usedto separate a particulate containing protein solution into threestreams:particulates trapped in surface treated rice hull ash pre-coat and bodyfeed,non-protein components bound to surface treated rice hull ash, andprotein components bound to and eluted off the surface treated rice hullash.

Example 14 Protein Binding Test

Materials

MilliQ H₂O

Protein solution (filtered catalase DFC)

50 mL Oak Ridge tubes

Sorval RC 5B Plus centrifuge with Sorval SA600 rotor

BCA Protein assay kit (Pierce)

Compat-Able Protein Assay Preparation Reagent Set (Pierce)

Pre-Diluted Protein Assay Standards, bovine serum albumin fraction V set(Pierce)

5 μm syringe filter (Sartoris, Minisart, #17594)

Procedures

-   1. 1 g of each silane treated rice hull ash Sample #54-67 was added    into a 50 mL Oak Ridge tube.-   2. 20 mL MilliQ H₂O was added to each tube.-   3. The contents of each tube was mixed by turning end-over-end at 8    rpm for 10 minutes at room temperature.-   4. Each tube was centrifuged at 16,000 rpm, 15° C. for 15 minutes.-   5. The supernatant was carefully removed using plastic transfer    pipettes.-   6. 1 part protein solution was diluted with 24 parts MilliQ H₂O    (Feed).-   7. 10 mL Feed was added to each tube (gave 10% w/v solid).-   8. Each tube was incubated at room temperature for 2 hours, turning    end-over-end at 8 rpm.-   9. Each tube was centrifuged at 16,000 rpm, 15° C. for 30 minutes.-   10. Each supernatant was filtered through a 0.45 μm syringe filters.-   11. The protein concentrations of Feed and filtered supernatants    (Step 10) were measured by BCA assay using microtiterplate protocol.    The results are shown as Table 12.

TABLE 12 Protein concentration ug/ml Sample # Silane Type Original160.66 Untreated RHA 98.75 54 trimethoxysilylpropyl-ethylenediamine,triacetic acid, trisodium 120.09 salt 55N-(triethoxysilylpropyl)-O-polyethylene oxide urethane 136.10 56Bis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane 112.02 57((chloromethyl)phenylethyl)trimethoxysilane 103.21 58N-(3-triethoxysilylpropyl)-gluconamide 81.02 593-mercaptopropyltriethoxysilane 73.43 60N-(triethoxysilylpropyl)-4-hydroxybutyramide 98.83 613-(triethoxysilyl)propylsuccinic anhydride 73.49 62Tris(3-trimethoxysilylpropyl)isocyanurate 81.41 632-Hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone 49.57 64Ureidopropyltrimethoxysilane 100.04 65 3-isocyanatopropyltriethoxysilane76.12 66 N-(3-trimethoxysilylpropyl)pyrrole 59.32 67Bis[(3-methyldimethoxysilyl)propyl]-polypropylene oxide 110.24Results

The protein concentrations of the Feed materials were decreased afterthe Feed materials were mixed with sample numbers 54-67, centrifuged,and filtered. The results indicate that silane-treated silica samplenumbers 54-67 and untreated RHA all bound proteins from the Feed proteinsolution.

Example 15 Test of Antimicrobial Activity (Bacillus subtilis)

Microorganism tested: Bacillus subtilis

Filter media tested: filter media samples 43, 44, 4 and FW12 (untreateddiatomaceous earth)

Protocol:

-   -   Bacillius subtilis fermentation broth was diluted in sterile PBS        to ˜10⁴ CFU/mL (1 OD≈5*10⁸ CFU/mL was used to estimate CFU/mL in        fermentation broth)    -   Use 0.5 g filter media/5 mL liquid (10% solid)

-   1. Serial dilutions (made in sterile 0.9% w/v NaCl) of the diluted    broth sample were plated on LA plates to determine actual CFU/mL    used. Plates were incubated over night at 34° C.

-   2. Filter media and diluted bacterial sample (or PBS control) were    mixed in a sterile 125 mL baffled flask for 2½ hours at 30° C., 200    rpm.

-   3. Liquid part of the treated samples (2) were plated on LA plates    (5 plates for each sample, one plate for control) and incubated    overnight at 34° C.

-   4. The plates were counted for bacteria.    Results:    The results are summarized in Table 13. By mixing the bacteria with    filter media samples 4 and 44, the CFUs were reduced, which    indicates that filter media samples 4 and 44 had anti-microbial    activity and killed the bacteria by contacting.

TABLE 13 Sample CFU/mL Diluted broth - start 6.53 * 10³ ± 2.47 * 10³Sample 43 + bacteria - mixing 1.04 * 10⁴ ± 1.50 * 10³ Sample 44 +bacteria - mixing 1.30 * 10² ± 3.00 * 10¹ Sample 4 + bacteria - mixingTFTC FW12 + bacteria - mixing 5.90 * 10⁴ ± 8.00 * 10³ Diluted brothsample - mixing 1.05 * 10³ ± 5.00 * 10¹Notes and Abbreviations:

-   -   PBS: Phosphate buffered saline (prevents cells from lysing due        to osmotic chock)    -   CFU: colony forming units (a measure of viable cells)    -   TFTC: Too Few To Count    -   The CFU/mL are reported as: Average±Difference (number of        plates) [the difference is between the average and the        observations farthest from the average].    -   Only plates with between 20-300 colonies were counted.

Example 16 Test of Antimicrobial Activity (Bacillus subtilis)

Microorganism tested: Bacillus subtilis

Filter Media tested: filter media samples 1, 4, 6, 44, and 45.

Protocol:

-   -   Bacillus subtilis fermentation broth was diluted in sterile PBS        to ˜10⁴ CFU/mL.    -   0.5 g filter media/5 mL liquid (10% solid) was used.

-   1. Serial dilutions (made in sterile 0.9% w/v NaCl) of the diluted    broth sample were plated on LA plates to determine actual CFU/mL    used. Plates were incubated over night at 34° C.

-   2. Filter media and diluted bacterial sample (15 mL liquid) were    mixed in a sterile 250 mL baffled flask. 2 flasks were used for each    filter media.    -   (A flask with PBS instead of bacterial sample was included for        the following filter media: Samples 1, 6 and 45)

-   3. The above was mixed for 2 hours at 30° C., 250 rpm.

-   4. Treated samples (the liquid part) were plated on LA plates (4 or    5 plates for each sample). Plates were incubated overnight at 34° C.

-   5. The plates were counted for bacteria.    Results:    The results are summarized in Table 14. By mixing the bacteria with    filter media samples 1, 4, 6, 44, and 45, the CFUs were    significantly reduced.

TABLE 14 Sample CFU/mL Diluted broth - start 3.45 * 10⁴ ± 4.50 * 10³Diluted broth - mixing 1.72 * 10⁴ ± 1.55 * 10³ Sample 1 A TFTC B TFTCSample 4 A TFTC B TFTC Sample 6 A TFTC B 1.00 * 10² ± 0.00 * 10⁰ Sample44 A 3.10 * 10² ± 9.00 * 10¹ B 6.00 * 10² Sample 45 A TFTC B TFTC

Example 17 Test of Antimicrobial Activity and Filtration (Lactobacillusbrevis)

Microorganism tested: Lactobacillus brevis

Filter media tested: Samples 4, 43, 45 & FW12.

Used 0.5 g filter media/5 mL culture (10% solid).

Protocol:

-   1. A Lactobacillus brevis overnight culture was diluted to ˜10⁵    CFU/mL (based on 1 OD₆₀₀≈2.7*10₈ CFU/mL) in two steps—the first    dilution was made in sterile Lactobacillus MRS broth, the second in    sterile PBS.-   2. Serial dilutions (in 0.9% w/v NaCl) of the culture were made    (second dilution).-   3. Diluted samples were plated on Lactobacillus MRS broth plates, to    determine actual starting CFU/mL.-   4. Filter media and diluted bacterial sample (10 mL liquid) were    mixed in a sterile 125 mL baffled flask, sealed with PARAFILM®, for    2 hours 15 minutes at room temperature on an orbit shaker (˜60 rpm).-   5. Serial dilutions (in 0.9% w/v NaCl) were made of treated sample    and plated on Lactobacillus MRS broth plates.-   6. Selected samples/dilutions of samples 4, 43 and 45 were filtered    through a 5 μm filter.-   7. The filtered samples were plated on Lactobacillus brevis broth    plates, and incubated in a candle jar at 30° C. for 2 days.-   8. The plates were counted.    Results:    The results are summarized in Table 15. CFUs were reduced by mixing    Samples 4, 43, and 45 with bacteria. CFUs were further reduced by    filtering the mixture through a 5 μm filter.

TABLE 15 Sample CFU/mL Lactobacillus brevis culture - start 1.05 * 10⁵ ±2.50 * 10³ Lactobacillus brevis culture - mixing 1.23 * 10⁵ ± 2.50 * 10³Sample 4 (mixing) 3.22 * 10⁴ ± 4.77 * 10³ Sample 43 (mixing) 3.43 * 10⁴± 5.67 * 10³ Sample 45 (mixing) 5.55 * 10² ± 4.50 * 10¹ FW12 (DE) 8.60 *10⁴ ± 4.75 * 10³ Filtered Sample 4 TFTC Filtered Sample 43 TFTC FilteredSample 45 TFTC

Example 18 Test of Antimicrobial Activity (E. coli)

Microorganism tested: E. coli (MG1655)

Filter media tested: FW12, samples 43, 1, 4, 6, 44 and 45.

Protocol:

0.5 g Filter Media/5 mL Feed (=10% solid).

-   1. An E. coli culture (not yet in stationary phase) was diluted to    ˜10⁵ CFU/mL (based on 1 OD₆₀₀≈5*10⁸ CFU/mL) in two steps—the first    dilution was made in sterile LB media, the second in sterile PBS    (this was the Feed).-   2. Serial dilutions (in 0.9% w/v NaCl) of the Feed were made.-   3. 100 μL of the diluted feed samples were plated on LA plates, to    determine the actual starting CFU/mL.-   4. Filter media and 10 mL feed were mixed in a sterile 125 mL    baffled flask for 2 hours at 25° C., 200 rpm (¾ inch stroke).-   5. Serial dilutions (in 0.9% w/v NaCl) of mixed samples were made    and 100 μl of each was plated on LA plates, and incubated overnight    at 30° C.-   6. Plates were counted.    Results:    The results are summarized in Table 16.

TABLE 16 Sample CFU/mL MG1655 - start 6.80 * 10⁴ ± 4.00 * 10³ MG1655 -mixing 5.35 * 10⁵ ± 2.50 * 10⁴ diatomaceous earth 2.28 * 10⁵ ± 1.72 *10⁵ Sample 43 9.05 * 10³ ± 5.50 * 10² Sample 1 1.28 * 10³ ± 2.45 * 10²Sample 4 1.73 * 10⁴ ± 2.03 * 10³ Sample 6 TFTC Sample 44 2.70 * 10³ ±1.23 * 10² Sample 45 5.20 * 10³ ± 2.00 * 10²

Example 19 Test of Antimicrobial Activity and Filtration (Lactobacillusbrevis)

Microorganism tested: Lactobacillus brevis type strain (ATCC#14869)

Filter media tested: Samples 43, 4, and 44

Protocol:

0.5 g Filter media/5 mL Feed (=10% solid)

-   1. A Lactobacillus brevis culture was diluted to ˜10⁵ CFU/mL (based    on 1 OD₆₀₀≈2.7*10⁸ CFU/mL) in two steps—the first dilution was made    in sterile Lactobacillus MRS broth, the second in sterile PBS (this    was the Feed).-   2. Serial dilutions (in 0.9% w/v NaCl) of the Feed were made.-   3. 100 μL of the diluted samples were plated on Lactobacillus MRS    broth plates, to determine the actual starting CFU/mL.-   4. Filter media and 5 mL Feed were mixed in a sterile 15 mL conical    tube for 2 hours at 25° C., 250 rpm (½ inch stroke).-   5. Serial dilutions (in 0.9% w/v NaCl) of mixed samples were made    and plated on Lactobacillus MRS broth plates (100 μl each).-   6. All samples were filtered through 5 μm syringe filter.-   7. Serial dilutions (in 0.9% w/v NaCl) of filtered samples were made    and plated on Lactobacillus MRS broth plates.-   8. Plates were counted in a candle jar at 30° C. for 2 days.-   9. Plates were counted.    Results:    The results are summarized in Table 17. CFUs were reduced by mixing    Samples 4, 43, and 44 with bacteria. CFUs were further reduced by    filtering the mixture through a 5 μm filter.

TABLE 17 Sample CFU/mL CFU/mL (filtered) ATCC#14869 - start 2.83 * 10⁴ ±4.67 * 10³ ATCC#14869 - 4.00 * 10⁴ ± 2.00 * 10³ 1.27 * 10⁴ ± 5.80 * 10²mixing Sample 43 4.55 * 10³ ± 3.50 * 10² 2.40 * 10³ ± 2.00 * 10² Sample4 1.95 * 10² ± 5.00 * 10⁰ TFTC Sample 44 8.10 * 10² ± 1.40 * 10² 5.50 *10¹ ± 5.00 * 10⁰

Example 20 Test of Antimicrobial Activity (Lactobacillus brevis)

Microorganism tested: Lactobacillus brevis

Filter media tested: Samples 48, 50, 51, and 52.

Protocol:

-   1. Lactobacillus brevis (gram positive) culture was streaked on MRS    agar and incubated anaerobically at 26° C. until growth was    sufficient.-   2. Working inoculum was prepared by diluting colonies from the MRS    plates into 0.1% peptone, targeting 5×104 cfu/mL.-   3. 0.5 g filter media was added to 10 mL inoculum in a 30 mL glass    tube (5%).-   4. The glass tube was sealed and incubated at room temperature for    30 minutes with mixing (8 inversions/minute).-   5. Serial dilutions of 1:10 were prepared in 0.9% NaCl and plated    with MRS agar, using the pour plate method to enumerate bacterial    population.-   6. Plates were incubated at 26° C., anaerobically (GasPak), until    growth was sufficient to count.-   7. Plates that had 20-200 colonies were counted. The Results are    summarized in Table 18.

Example 21 Test of Antimicrobial Activity (Acetobacter pasteurianus(Gram Negative))

Microorganism tested: Acetobacter pasteurianus (gram negative)

Filter media tested: Samples 48, 50, 51, and 52.

Protocol:

-   1. Acetobacter pasteurianus (gram negative) culture was streaked    onto MRS agar and incubated aerobically at 27° C. until growth was    sufficient.-   2. Culture was stocked by adding 1 mL loop of agar plate colonies to    99 mL of MRS broth and incubated at 27° C.-   3. Working inoculum was made by diluting an aliquot of the MRS stock    culture into either phosphate buffered saline (PBS) or 0.1% peptone.-   4. 0.5 g of filter media was added to 10 mL inoculum in a 30 mL    glass tube.-   5. The glass tube was sealed and incubated at room temperature for    30 minutes with mixing (8 inversions/minute).-   6. Serial dilutions of 1:10 were performed in 0.1% peptone and    plated with MRS agar, using the pour plate method to enumerate    bacterial population.-   7. Plates were counted at 27° C., aerobically, until growth was    sufficient to count.-   8. Plates that had 20-200 colonies were counted. The Results are    summarized in Table 18.

Example 22 Test of Antimicrobial Activity (Saccharomyces diastaticus(Yeast))

Microorganism tested: Saccharomyces diastaticus (yeast)

Filter media tested: Samples 48, 50, and 51.

Protocol:

-   1. Saccharomyces diastaticus (yeast) culture was streaked onto YM    agar and incubated aerobically at 30° C. until growth was    sufficient.-   2. Working inoculum was prepared by diluting colonies from the YM    plates into phosphate buffered saline (PBS), targeting 3×104 cfu/mL.-   3. 0.5 g to filter media was added to 10 mL inoculum in a 30 mL    glass tube.-   4. The glass tube was sealed and incubated at room temperature for    30 minutes with mixing (8 inversions/minute).-   5. Serial dilutions of 1:10 were performed in 0.9% NaCl and plated    with MRS agar, using the pour plate method to enumerate bacterial    population.-   6. Plates were incubated at 30° C., aerobically, until growth was    sufficient to count.-   7. Plates that had 20-200 colonies were counted. The Results are    summarized in Table 18.

TABLE 18 Lactobacillus Acetobacter Saccharomyces Brevis, gramspasteurinus, distaticus, Sample positive (+) gram negative (−) yeast No.Treatment Silica Type % Reduction % Reduction % Reduction 483-(N-styrylmethyl-2- RiceSil 100 100% 18% 41% aminoethylamino)-propyltrimethoxy- silane hydrochloride 51 3-trihydroxysilylpropyl-RiceSil 100 20% 10% 33% methyl phosphonate, sodium salt 50N-(2-Aminoethyl)-3- RiceSil 100 90% 20% 3% aminopropyltrimethoxy- silaneGlycidyltrimethyl- ammonium chloride 52 N- RiceSil 100 100% 90%Octadecyldimethyl(3- Trimethoxysilylpropyl) ammonium chloride

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications could be made without departing from the scope of theinvention.

1. Silane-treated silica filter media prepared by reacting the surfaceactive group of silica filter media with silanes selected from the groupconsisting of 3-aminopropyltrimethoxysilane andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane;3-trihydrosilylpropylmethylphosphonate, sodium salt andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane;N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride and(3-glycidoxypropyl)trimethoxysilane;3-trihydrosilylpropylmethylphosphonate, sodium salt andbis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane;3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilanehydrochloride and N-(triethoxysilylpropyl)-O-polyethylene oxideurethane; 2-(trimethoxysilylethyl)pyridine andN-(3-triethoxysilylpropyl)-gluconamide;N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride andN-(3-triethoxysilylpropyl)-gluconamide;N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride and2-hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone;3-mercaptopropyltriethoxysilane andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane;3-(triethoxysilyl)propylsuccinic anhydride andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane;trimethoxysilylpropyl-ethylenediamine, triacetic acid, trisodium saltand N-(triethoxysilylpropyl)-O-polyethylene oxide urethane;2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane; and2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane andbis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane, wherein the silicafilter media are rice hull ash, oat hull ash or diamataceous earth. 2.The silane-treated silica filter media according to claim 1, whereinsaid silanes are 3-aminopropyltrimethoxysilane andN-(triethoxysilylpropyl)-O-polyethylene oxide urethane.
 3. Thesilane-treated silica filter media according to claim 1, wherein saidsilanes are 3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilanehydrochloride and N-(triethoxysilylpropyl)-O-polyethylene oxideurethane.
 4. Silane-treated silica filter media prepared by reacting thesurface active group of silica filter media with one or more silanesselected from the group consisting of2-hydroxy-4-(3-triethoxysilylpropoxy)-diphenlyketone,2-(trimethoxysilylethyl)pyridine, N-(triethoxysilypropyl)-O-polyethyleneoxide urethane, 3-(trimethoxysilyl)propyl methacrylate,3-(triethoxysilyl)propylsuccinic anhydride,tris(3-trimethoxysilylpropyl)isocyanurate,bis[(3-methyldimethoxysilyl)propyl]-polypropylene oxide,2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane,N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole,2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane,trimethoxysilylpropylisothiouronium chloride,N-(3-triethoxysilylpropyl)-gluconamide,N-(triethoxysilylpropyl)-4-hydroxy butyramide, andO-(propargyloxy)-N-(triethoxysilylpropyl)urethane, wherein the silicafilter media are rice hull ash, oat hull ash, or diamotaceous earth. 5.The silane-treated silica filter media according to claim 4, whereinsaid silane is 2-hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone. 6.The silane-treated silica filter media according to claim 4, whereinsaid silane is N-(triethoxysilylpropyl)-O-polyethylene oxide urethane.7. The silane-treated silica filter media according to claim 4, whereinsaid silane is 3-(triethoxysilyl)propylsuccinic anhydride.
 8. Thesilane-treated silica filter media according to claim 4, wherein saidsilane is tris(3-trimethoxysilylpropyl)isocyanurate.
 9. Thesilane-treated silica filter media according to claim 4, wherein saidsilane is bis[(3-methyldimethoxysilyl)propyl]-polypropylene oxide. 10.The silane-treated silica filter media according to claim 4, whereinsaid silane is N-(3-triethoxysilylpropyl)-gluconamide.
 11. Thesilane-treated silica filter media according to claim 4, wherein saidsilane is N-(triethoxysilylpropyl)-4-hydroxybutyramide.
 12. Thesilane-treated silica filter media according to claim 4, wherein saidsilane is 2-(trimethoxysilylethyl)pyridine.
 13. The silane-treatedsilica filter media according to claim 4, wherein said silane is3-(trimethoxysilyl)propyl methacrylate.
 14. The silane-treated silicafilter media according to claim 4, wherein said silane is2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane.
 15. The silane-treatedsilica filter media according to claim 5, wherein said silane isN-(3-triethoxysilylpropyl)-4,5-dihydroimidazole.
 16. The silane-treatedsilica filter media according to claim 4, wherein said silane is2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane.
 17. The silane-treatedsilica filler media according to claim 4, wherein said silane istrimethoxysilylpropylisothiouronium chloride.
 18. The silane-treatedsilica filter media according to claim 4, wherein said silane isO-(propargyloxy)-N-(triethoxysilylpropyl)urethane.